JP2018143253A - Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To address a pressing need for alternative and robust systems and techniques for sequence targeting with a wide array of applications and provide related advantages.SOLUTION: The invention provides engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are compositions and methods related to components of a CRISPR complex particularly comprising a Cas ortholog enzyme.SELECTED DRAWING: Figure 1

Description

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE This application is a US Provisional Patent Application No. 61 / 836,101 filed Jun. 17, 2013, entitled ENGINEERING AND OPTIMIZATION OF IMPROVED SYSTEMS, METHODS AND ENZYME COMPOSITIONS FORSEQUENION Claim priority. This application is based on US Provisional Patent Application Nos. 61 / 758,468; 61 / 769,046; 61 / 802,174; 61 / 806,375. Claims 61 / 814,263; 61 / 819,803 and 61 / 828,130, the titles ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND, respectively. COMPOSIONS FOR SEQUENCE MANIPULATION, January 30, 2013; February 25, 2013, March 15, 2013; March 28, 2013, April 20, 2013; May 6, 2013 and It was filed on May 28, 2013. This application also claims the priority of US Provisional Patent Application Nos. 61 / 736,527 and 61 / 748,427, both of which are entitled SYSTEMS METHODS AND COMPOSTIONS FOR SEQUENCE MANIPULATION, 2012 each. It was filed on December 12, 2013 and January 2, 2013. The priority of US Provisional Patent Application Nos. 61 / 791,409 and 61 / 835,931 filed on March 15, 2013 and June 17, 2013, respectively, is also claimed.
U.S. Provisional Patent Application Nos. 61 / 836,127, 61 / 835,936, 61 / 836,080 and 61, filed on June 17, 2013, respectively. No. 8,836,123 and 61 / 835,973.

  The above applications, as well as all documents cited in those applications or during their examination ("application citations") and all documents cited or referenced in the application citations, as well as cited or All references referred to ("citations herein"), and all references cited or referenced in the references cited herein, are hereby or in any reference incorporated herein by reference. Together with any manufacturer's instructions, instructions, product specifications, and product sheets for any of the products listed, it is incorporated herein by reference and can be used in the practice of the present invention. More specifically, all references are incorporated by reference to the extent that each individual document is shown to be specifically incorporated by reference.

  The present invention generally relates to a system used for the control of gene expression including clustered regularly interspersed short palindromic repeats (CRISPR) and its components, eg, genomic perturbation or gene editing. , Engineering and optimization of methods and compositions.

DESCRIPTION OF FEDERALLY SPONSORED RESEARCH This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) funded by the National Institutes of Health. The US government has certain rights in this invention.

  Recent advances in genome sequencing techniques and analytical methods have significantly accelerated the ability to classify and map genetic factors associated with a diverse range of biological functions and diseases. Accurate genomic targeting technology enables systematic reverse engineering of causal genetic mutations by allowing selective perturbation of individual genetic elements and advances synthetic biology, biotechnology and pharmaceutical applications Is needed for. Genome editing techniques, such as designer zinc fingers, transcriptional activator-like effectors (TALE), or generation of genomic perturbations targeted to homing meganucleases, are inexpensive, easy to set up, scalable, true There remains a need for new genome engineering techniques that are easy to target multiple positions in the nuclear genome.

International Publication No. 97/03211 Pamphlet

  The CRISPR / Cas or CRISPR-Cas system (both terms are used interchangeably throughout this application) does not require the production of a custom protein to target specific sequences, but a single Cas enzyme. Programming with short RNA molecules allows specific DNA targets to be recognized, in other words, Cas enzymes can be recruited to specific DNA targets using the short RNA molecules. The addition of the CRISPR-Cas system to the genome sequencing technology and analytical method repertoire significantly simplifies the methodology and accelerates the ability to classify and map genetic factors associated with a wide range of biological functions and diseases. The In order to effectively utilize the CRISPR-Cas system for genome editing without adverse effects, it is important to understand the engineering and optimization aspects of those genomic engineering tools that are aspects of the claimed invention. is there.

  Therefore, there is an urgent need for alternative and robust systems and techniques for versatile sequence targeting. Aspects of the present invention address this need and provide related advantages. An exemplary CRISPR complex includes a CRISPR enzyme complexed with a guide sequence that is hybridized to a target sequence within a target polynucleotide, the CRISPR enzyme including, but not limited to, Corynebacter. ), Stellala, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Lactobacillus Mycoplasma), Bacteroides, Flaviivola, Flavova The genus Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburbium, Roseburvula Cas orthologs of the genera including (Staphylococcus), Nitratifractor, Mycoplasma, and Campylobacter, for example, Cas9 ortholog. The guide sequence is then linked to a tracr mate sequence that hybridizes to the tracr sequence.

  In one aspect, the present invention provides a method of using one or more elements of the CRISPR system. The CRISPR complex of the present invention provides an effective means for modifying the target polynucleotide. The CRISPR complexes of the present invention have broad utility, for example, modification of target polynucleotides in a large number of cell types (eg, deletion, insertion, translocation, inactivation, activation, repression, methylation). Change, movement of specified part). Thus, the CRISPR complexes of the present invention have a wide range of applications in, for example, gene or genome editing, gene regulation, gene therapy, drug delivery, drug screening, disease diagnosis, and prognosis. In a preferred embodiment of the present invention, the CRISPR complex includes, but is not limited to, Corynebacter, Suterella, Legionella, Treponema, Filifactor. Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivibola, Flavivivola, Flavivivola Sphaerochaeta), Azospirillum (Azospirillum), Gluconacetobacter, Neisseria, Roseburia, Parvibacrum, Staphylococci, Staphylococci Cas enzymes from the genera, including (Mycoplasma) and Campylobacter, preferably the Cas9 ortholog.

  Aspects of the invention relate to a CRISPR enzyme having an optimization function. With respect to the CRISPR enzyme, which is a Cas enzyme, a preferred embodiment of the present invention relates to a Cas9 ortholog with improved target specificity in the CRISPR-Cas9 system. This includes, but is not limited to, the design and preparation of guide RNA with optimal activity, the selection of a Cas9 enzyme of a defined length, the truncation of Cas9 enzyme (by truncating a nucleic acid molecule encoding Cas9). , Shortening the length of Cas9 relative to the corresponding wild-type Cas9 enzyme), as well as chimeric Cas9 enzyme (where different parts of the enzyme are swapped or exchanged between different orthologs to obtain a chimeric enzyme with a regulated specificity) Can be achieved by the following approaches: Aspects of the invention also provide a method for improving the target specificity of Cas9 ortholog enzyme or a method for designing a CRISPR-Cas9 system comprising designing or preparing a guide RNA with optimal activity and / or corresponding wild-type Selecting or preparing a Cas9 orthologous enzyme having a size or length smaller than that of type Cas9 (therefore, the coding sequence in the delivery vector is shorter than the corresponding wild-type Cas9 and therefore the nucleic acid delivery vector encoding it) And / or producing a chimeric Cas9 enzyme.

  Also provided is the use of the sequences, vectors, enzymes or systems of the invention in medicine. Also provided are sequences, vectors, enzymes or systems of the invention for use in gene or genome editing. Also provided is the use of a sequence, vector, enzyme or system of the present invention in the manufacture of a medicament for gene or genome editing, eg, therapy by gene or genome editing. Also provided are sequences, vectors, enzymes or systems of the invention for use in therapy.

  In additional aspects of the invention, a CRISPR enzyme, eg, a Cas9 enzyme, may contain one or more mutations, has or does not have a fusion with a functional domain, or is operably linked thereto. It can be used as a generic DNA binding protein. The mutation can be an artificially introduced mutation, which includes, but is not limited to, one or more mutations in the catalytic domain. Examples of catalytic domains for the Cas9 enzyme include, but are not limited to, RuvCI, RuvCII, RuvCIII and HNH domains. Preferred examples of suitable mutations are catalytic residues in the N-terminal RuvCI domain of Cas9 or catalytic residues in the internal HNH domain. In some embodiments, Cas9 is (or is derived from) Streptococcus pyogenes Cas9 (SpCas9). In such embodiments, preferred mutations are the corresponding positions in SpCas9 at positions 10,762, 840, 854, 863 and / or 986 or other Cas9 orthologs with respect to the position numbering of SpCas9 (eg, standard sequences). In any or all of the comparison tools, eg MegAlign by ClustalW or Lasergene 10 suite). In particular, any or all of the following mutations in SpCas9: D10A, E762A, H840A, N854A, N863A and / or D986A are preferred; conservative substitutions for any of the substituted amino acids are also envisioned. Also preferred are identical mutations (or conservative substitutions of those mutations) at corresponding positions with respect to the position numbering of SpCas9 in other Cas9 orthologs. D10 and H840 in SpCas9 are particularly preferred. However, in other Cas9, residues corresponding to SpCas9D10 and H840 are also preferred. These are converted to catalytically ineffective mutants that are useful for global DNA binding because they provide nickase activity when mutated alone, and when both mutations are present. Therefore, it is advantageous. Additional mutations have been identified and characterized. Other aspects of the invention include, but are not limited to, transcriptional activators, transcriptional repressors, recombinases, transposases, histone remodelers, DNA methyltransferases, cryptochromes, light-inducible / regulatory domains or It relates to a mutant Cas9 enzyme that is fused or operably linked to a domain that includes a chemically inducible / regulatory domain.

  Further aspects of the invention provide chimeric Cas9 proteins and methods for producing chimeric Cas9 proteins. A chimeric Cas9 protein is a protein that contains fragments originating from different Cas9 orthologs. For example, a Cas9 chimeric protein obtained by fusing the N-terminus of a first Cas9 ortholog with the C-terminus of a second Cas9 ortholog can be generated. These chimeric Cas9 proteins may have a specificity or efficiency that is higher than the original specificity or efficiency of any of the individual Cas9 enzymes from which the chimeric protein is produced. These chimeric proteins can also contain one or more mutations or can be linked to one or more functional domains. Accordingly, an aspect of the invention relates to a chimeric Cas enzyme comprising one or more fragments from a first Cas ortholog and one or more fragments from a second Cas ortholog. In one embodiment of the invention, the one or more fragments of the first or second Cas ortholog are from the C or N terminus of the first or second Cas ortholog. In further embodiments, the first or second Cas orthologs are Corynebacter, Stutterella, Legionella, Treponema, Filifactor, Eubacterium. (Eubacterium), Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, et al, S (Azospi illum), Gluconacetobacter genus, Neisseria genus, Roseburia genus, Parbibaculum genus, Staphylococcus genus Staphylococcus genus, Nitralati fractor genus citrus Mycoplasma) and a genus belonging to the group consisting of Campylobacter.

  In a further embodiment, the present invention provides a method of generating a mutant component of a CRISPR complex comprising a Cas enzyme, eg, a Cas9 ortholog. Mutant components can include, but are not limited to, mutant tracRNA and tracr mate sequences or mutant chimeric guide sequences that allow for improved performance of those RNAs in the cell. Also provided is the use of the present composition or enzyme in the preparation of a medicament for modification of a target sequence.

In yet a further aspect, the present invention provides:
A) -I. A CRISPR-Cas chimeric RNA (chiRNA) polynucleotide sequence comprising:
(A) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell; (b) a tracr mate sequence; and (c) a polynucleotide sequence comprising a tracr sequence; and II. A polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences;
(A), (b) and (c) are arranged in a 5 ′ to 3 ′ orientation;
When transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs the sequence specific binding of the CRISPR complex to the target sequence;
The CRISPR complex includes (1) a guide sequence that is hybridized to a target sequence, and (2) a CRISPR enzyme that is complexed with a tracr mate sequence that is hybridized to a tracr sequence, The nucleotide sequence may comprise a non-naturally occurring or engineered composition that is DNA or RNA or (B) I.I. A polynucleotide comprising:
(A) a guide sequence capable of hybridizing to a target sequence in a prokaryotic cell, and (b) a polynucleotide comprising at least one or more tracr mate sequences,
II. A polynucleotide sequence encoding a CRISPR enzyme, and III. comprising a polynucleotide sequence comprising a tracr sequence;
When transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs the sequence-specific target binding of the CRISPR complex to the target sequence;
The CRISPR complex includes (1) a guide sequence that is hybridized to a target sequence, and (2) a CRISPR enzyme that is complexed with a tracr mate sequence that is hybridized to a tracr sequence, The nucleotide sequence is DNA or RNA,
CRISPR enzymes can be selected from Corynebacter, Suterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Streptococcus, Streptococcus, Streptococcus Genus (Lactobacillus), Mycoplasma (Mycoplasma), Bacteroides (Bacteroides), Flavivibola (Flavivovola), Flavobacterium (Sphaerochaetalu), Azosporarum luconacetobacter, Neisseria, Roseburia, Parvivaculum, Staphylococcus, Nitratifacta, Nitratifacta, Mytrafactor Compositions and methods relating to non-naturally occurring or engineered compositions that are Cas9 orthologs of the genus belonging to the group consisting of:

  In a still further aspect, the present invention provides (A) I.I. A first regulatory element operably linked to a CRISPR-Cas based chimeric RNA (chiRNA) polynucleotide sequence (the polynucleotide sequence is (a) a guide sequence that can hybridize to a target sequence in a eukaryotic cell; b) a tracr mate sequence, and (c) a tracr sequence), and II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are 5 Arranged in a 'to 3' orientation, components I and II are on the same or different vectors of the system, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence to the target sequence. Directing sequence-specific binding of the CRISPR complex, wherein the CRISPR complex is complexed with (1) a guide sequence that is hybridized to the target sequence, and (2) a tracr mate sequence that is hybridized to the tracr sequence. CRISPR enzyme, which is a CRISPR enzyme, such as Corynebacter, Stella lla), Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Lactobacillus, Lactobacillus, (Bacteroides), Flavivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacteria N, Gluconacetobacteria N, Seburia), Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, and Camphorobacter (Campylobacter) belonging to the group 9 belonging to the genus C belonging to the genus C. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors or (B) (A) a guide sequence capable of hybridizing to a target sequence in a prokaryotic cell, and (b) a first regulatory element operably linked to at least one or more tracr mate sequences, II. A second regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, and III. contains a third regulatory element operably linked to the tracr sequence, components I, II, and III are on the same or different vectors of the system, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence And the guide sequence directs the sequence specific binding of the CRISPR complex to the target sequence, the CRISPR complex is hybridized to (1) a guide sequence that is hybridized to the target sequence, and (2) a tracr sequence. A CRISPR enzyme complexed with a tracr mate sequence, wherein the CRISPR enzyme comprises: Corynebacter, Suterella, Legionella, Treponema, Filifama tor), Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivivore, Flavivivola Genus (Sphaerochaeta), Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvivoculocus, S. A Cas9 ortholog of the genus belonging to the group consisting of Nitratifractor, Mycoplasma, and Campylobacter, including a vector system comprising one or more vectors to which at least one of the following criteria applies: A non-naturally occurring or engineered composition is provided.

The criteria are as follows and any number thereof may apply, preferably one or more, preferably two or more, preferably three or more, four or more, or five or more, or all It is recognized that you get:
A CRISPR enzyme having a defined size is selected and is at least 500 amino acids, at least 800-899 amino acids, at least 900-999 amino acids, at least 1000-1099 amino acids, at least 1100-1199 amino acids, at least 1200-1299 amino acids, at least 1300-1399 Having an amino acid length of at least 1400-1499 amino acids, at least 1500-1599 amino acids, at least 1600-1699 amino acids or at least 2000 amino acids;
-And / or the CRISPR enzyme is truncated compared to the corresponding wild-type CRISPR enzyme;
-And / or the CRISPR enzyme is a nuclease that directs the cleavage of both strands in the location of the target sequence, or the CRISPR enzyme is a nickase that directs the cleavage of one strand in the location of the target sequence;
-And / or the guide sequence comprises at least 10, at least 15 or at least 20 nucleotides;
-And / or the CRISPR enzyme is codon optimized or codon optimized for expression in eukaryotic cells;
-And / or the CRISPR enzyme comprises one or more mutations;
-And / or the CRISPR enzyme comprises a chimeric CRISPR enzyme;
-And / or the CRISPR enzyme has one or more other attributes as discussed herein.

  In some embodiments, the CRISPR enzyme is truncated as compared to the wild type CRISPR enzyme, or the CRISPR enzyme is at least 500 amino acids, at least 800-899 amino acids, at least 900-999 amino acids, at least 1000-1099 amino acids. , At least 1100-1199 amino acids, at least 1200-1299 amino acids, at least 1300-1399 amino acids, at least 1400-1499 amino acids, at least 1500-1599 amino acids, at least 1600-1699 amino acids, or at least 2000 amino acids. In a preferred embodiment, the CRISPR enzyme is a Cas enzyme, such as a Cas9 ortholog.

  In some embodiments, the CRISPR enzyme is a nuclease that directs cleavage of both strands in the target sequence location, or the CRISPR enzyme is a nickase that directs cleavage of one strand in the target sequence location. is there. In a further embodiment, the CRISPR enzyme is a catalytically ineffective mutant that is a global DNA binding protein. In a preferred embodiment, the CRISPR enzyme is a Cas enzyme, such as a Cas9 ortholog.

  In some embodiments, the guide sequence comprises at least 15 nucleotides. In some embodiments, the CRISPR enzyme is codon optimized or codon optimized for expression in eukaryotic cells. In some embodiments, the CRISPR enzyme comprises one or more mutations. In some embodiments, the CRISPR enzyme comprises a chimeric CRISPR enzyme. In some embodiments, the CRISPR enzyme has one or more other attributes as discussed herein. In a preferred embodiment, the CRISPR enzyme is a Cas enzyme, such as a Cas9 ortholog.

  In certain embodiments, the CRISPR enzyme comprises one or more mutations. One or more mutations may be present in a particular domain of the enzyme. In preferred embodiments, one or more mutations may be present in the catalytic domain. In a further preferred embodiment, the catalytic domain is a RuvCI, RuvCII, RuvCIII or HNH domain. In a more preferred embodiment, the one or more mutations are present in the RuvC1 or HNH domain of the CRISPR enzyme. In a further preferred embodiment, the CRISPR enzyme is a Cas enzyme, such as a Cas9 ortholog, and the mutation corresponds to, but is not limited to, D10A, E762A, H840A, N854A, N863A or D986A with respect to the position numbering of SpCas9. Mutations that may be at one or the positions mentioned and / or are discussed elsewhere herein. In some embodiments, the CRISPR enzyme has one or more mutations in a particular domain of the enzyme, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence to the target sequence. Directed to sequence-specific binding of the CRISPR complex, the enzyme further comprising a functional domain. Functional domains include but are not limited to transcriptional activators, transcriptional repressors, recombinases, transposases, histone remodelers, DNA methyltransferases, cryptochromes, light-inducible / regulatory domains or chemically-inducible / Regulatory domains.

  In some embodiments, the functional domain is the transcriptional activator domain VP64. In some embodiments, the functional domain is the transcriptional repressor domain KRAB. In some embodiments, the transcriptional repressor domain is a SID or a concatemer of SIDs (ie, SID4X). In some embodiments, epigenetic modifying enzymes, such as histone modified proteins or epigenetic chromatin modified proteins are provided. In some embodiments, an activator domain is provided that can be a P65 activator domain.

A further aspect of the invention includes a method of modifying two or more target genomic loci. In a preferred embodiment of the present invention, two or more genomic loci are modulated separately by utilizing one or more CRISPR enzymes, eg, two or more Cas9 orthologs, each ortholog being one It is operably linked to these functional domains. In one aspect, the present invention provides a method of modifying two or more genomic loci in a eukaryotic cell. Accordingly, an aspect of the present invention is a method for modulating the expression of two or more target genomic loci in an organism, comprising:
I. A first regulatory element operatively linked to a first CRISPR-Cas chimeric RNA (chiRNA) polynucleotide sequence (the first polynucleotide sequence is
(Ii) a first guide sequence capable of hybridizing to a first target sequence at a first genomic locus in a cell of the organism; (ii) a first tracr mate sequence; and (iii) a first tracr sequence. And II. A second regulatory element operatively linked to a second CRISPR-Cas based chimeric RNA (chiRNA) polynucleotide sequence (the second polynucleotide sequence is
(I) a second guide sequence capable of hybridizing to a second target sequence at a second genomic locus in a cell of the organism;
(Ii) including a second tracr mate sequence, and (iii) a second tracr sequence), and III. A third regulatory element operably linked to an enzyme coding sequence encoding a first CRISPR enzyme comprising at least one or more nuclear localization sequences and operably linked to a first functional domain ,
IV. A fourth regulatory element operably linked to an enzyme coding sequence encoding a second CRISPR enzyme comprising at least one or more nuclear localization sequences and operably linked to a second functional domain Wherein (i), (ii) and (iii) of I and II are arranged in a 5 ′ to 3 ′ orientation and components I, II, III and IV are on the same or different vectors of the system Yes, when transcribed, hybridizes to the respective tracr mate sequence g and gcr sequence, and the first and second guide sequences of the first and second CRISPR complexes to the first and second target sequences Directed for sequence-specific binding, the CRISPR complex comprises (1) a guide sequence that is hybridized to the target sequence, and (2) a tracr mate that is hybridized to the tracr sequence. A CRISPR enzyme complexed with the sequence, wherein expression of the CRISPR enzyme provides manipulation of the target sequence, the first and second CRISPR enzymes each include two or more mutations, The second CRISPR enzymes are Corynebacter, Stutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Streptococcus, Streptococcus ), Lactobacillus, Mycoplasma, Bacteroides, Flaviiv ola), Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria ur, V, Roseburi ur R A vector system comprising one or more vectors that are Cas9 orthologs of the genus belonging to the group consisting of Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter Non-natural or engineering Delivering a modified composition, wherein the first genomic locus is modulated by the activity of the first functional domain and the second genomic locus is modulated by the activity of the second functional domain Provide a method. In further embodiments, the first functional domain is a transcriptional activator, transcriptional repressor, recombinase, transposase, histone remodeler, DNA methyltransferase, cryptochrome and light-inducible / regulatory domain or chemically-inducible / Selected from the group consisting of regulatory domains. In a further embodiment, the second functional domain is a transcriptional activator, transcriptional repressor, recombinase, transposase, histone remodeler, DNA methyltransferase, cryptochrome and light-inducible / regulatory domain or chemically-inducible / Selected from the group consisting of regulatory domains. In a preferred embodiment, the first or second CRISPR enzyme is Sterella wadsworthensis Cas9, Filifactor alocis Cas9, Lactobacillus johnsonoxy9 lari) Cas9, Corynebacter diptheliae Cas9, Pervivacumum lavamentivorans Cas9, Mycoplasma galepticum 9 Staphylococcus aureus (Staphylococcus aureus subsubspecies Aureus) Cas9, Legionella pneumophila (Legionella pneumophila) Paris Cas9, Treponema denticola (Treponema denticola) Cas9, Staphylococcus shoe de Inter-media scan (Staphylococcus pseudintermedius) Cas9, Neisseria cinerea (Neisseria cinerea) Cas9.

  In some embodiments, the CRISPR enzyme is a type I, II or III CRISPR enzyme, preferably a type II CRISPR enzyme. The type II CRISPR enzyme can be any Cas enzyme. The Cas enzyme can be identified as Cas9. This is because it can refer to a general class of enzymes that share homology with the largest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from or derived from SpCas9 or Staphylococcus aureus subspecies Aureus SaCas9. Originating is based on the wild-type enzyme in the sense that the majority of the derived enzyme has a high degree of sequence homology with the wild-type enzyme, but this is not the case with some techniques described herein. It means that it has been mutated (modified).

  It will be appreciated that the terms Cas and CRISPR enzymes are commonly used interchangeably herein, unless otherwise apparent. As noted above, much of the residue numbering used herein refers to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, the present invention is not limited to other microbial species, such as Corynebacter, Stutterella, Legionella, Treponema, Filifactor, Eubacterium. ), Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivivala, Flavobacterium, Flavobacterium, Flavobacterum ), Gluconacetobacter, Neisseria, Roseburia, Parvibacrum, Staphylococcus, Nitratifurac, or Nitratifras It is recognized that it contains more Cas9 from those belonging to the genus Campylobacter, such as SpCas9, SaCas9, St1Cas9, St3Cas9, etc. St is a Streptococcus thermophilus.

  In this case, examples of codon optimized sequences optimized for humans (ie optimized for expression in humans) are provided herein (see SaCas9 human codon optimized sequences). While this is preferred, it will be appreciated that other examples are possible and codon optimization for the host species is known.

  Further aspects of the invention include improved cleavage specificity, optimized tracr sequences, optimized chimeric guide RNA, co-fold structure of tracrRNA and tracr mate sequences, secondary structure of tracrRNA. It relates to a stabilization, a trcrRNA with a shortened region of base pairing, a tracrRNA with a fusion RNA element, a simplified cloning and delivery, a reduced toxicity and / or inducible system. Another aspect of the invention relates to the stabilization of chimeric RNAs and / or guide sequences and / or their portions of the CRISPR complex, wherein the CRISPR enzyme is a CRISPR ortholog, the chimeric RNA, and / or the guide sequence and / or Some of them are stabilized by synthetic or chemically modified nucleotides (eg, LNA / BNA: thiol modified, 2 ′ / 3′-OH cross-linked modified) and modified to become degradation / hydrolysis resistant A stability element was added.

  The present invention, in certain embodiments, is a method of modifying an organism or non-human organism by manipulating a target sequence in a genomic locus of interest, operable for expression of the composition discussed herein. Further included is a method comprising delivering a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors encoding in Preferably, the vector is a viral vector, such as a lentivirus or baculovirus, or preferably an adenovirus / adeno-associated virus vector, but other delivery means are known (eg, yeast systems, microvesicles, genes Means for attaching the gun / vector to the gold nanoparticles).

  Various delivery means are described herein and are further discussed in this section.

  Viral delivery: CRISPR enzymes, such as Cas9 and / or any of the present RNAs, such as guide RNA, using adeno-associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof Can be delivered. Cas9 and one or more guide RNAs can be packaged in one or more viral vectors. In some embodiments, the viral vector is delivered to the tissue of interest, for example, by intramuscular injection, while in other cases viral delivery is intravenous, transdermal, intranasal, oral, mucosal, or other Depending on the delivery method. Such delivery may be by a single dose or by multiple doses. One skilled in the art will recognize that the actual dosage delivered herein is the choice of vector, target cell, organism or tissue, the general condition of the subject being treated, the degree of transformation / modification required, the route of administration, the method of administration, It will be appreciated that it can vary greatly depending on various factors such as the type of transformation / modification required.

  Such dosages are, for example, carriers (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), diluents, pharmaceutically acceptable carriers (Eg, phosphate buffered saline), pharmaceutically acceptable excipients, adjuvants that enhance antigenicity, immunostimulatory compounds or molecules, and / or other compounds known in the art Can do. An adjuvant in this specification is a suspension of mineral (alum, aluminum hydroxide, aluminum phosphate) to which an antigen is adsorbed; or a water-in-oil emulsion (MF-59, Freund's) in which an antigen solution is emulsified in oil. Incomplete adjuvants (sometimes containing dead mycobacteria (Freund's complete adjuvant)) can be included to further enhance antigenicity (inhibiting antigen degradation and / or causing macrophage influx). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and, for example, immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides. Such dosage formulation is readily ascertainable by one skilled in the art. The dosage is one or more pharmaceutically acceptable salts, for example mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate; and acetate, propionate, malonate Further, an organic acid salt such as benzoate can be further contained. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavoring agents, coloring agents, microspheres, polymers, suspending agents and the like may also be present herein. In addition, one or more other conventional pharmaceutical ingredients such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers Etc. may also be present, especially when the dosage form is in a reconfigurable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, paraben, ethyl vanillin, glycerin, phenol, Parachlorophenol, gelatin, albumin and combinations thereof. For a detailed discussion of pharmaceutically acceptable excipients, reference may be made to REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), incorporated herein by reference.

In certain embodiments herein, delivery is via adenovirus, which can be a single booster dose containing an adenoviral vector of at least 1 × 10 ⁵ particles (particle unit, also referred to as pu). In certain embodiments herein, the dose is preferably at least about 1 × 10 ⁶ particles (eg, about 1 × 10 ⁶ to 1 × 10 ¹² particles), more preferably at least about 1 × 10 ⁷ particles, more preferably Is at least about 1 × 10 ⁸ particles (eg, about 1 × 10 ⁸ to 1 × 10 ¹¹ particles or about 1 × 10 ⁸ to 1 × 10 ¹² particles), and most preferably at least about 1 × 10 ⁸ particles (eg, About 1 * × 10 ⁹ to 1 × 10 ¹⁰ particles or about 1 × 10 ⁹ to 1 × 10 ¹² particles), or even at least about 1 × 10 ¹⁰ particles (eg, about 1 × 10 ¹⁰ to 1 × 10 ¹² particles). ) Adenovirus vector. Alternatively, the dose is about 1 × 10 ¹⁴ particles or less, preferably about 1 × 10 ¹³ particles or less, even more preferably about 1 × 10 ¹² particles or less, even more preferably about 1 × 10 ¹¹ particles or less, and most preferably Comprises about 1 × 10 ¹⁰ particles or less (eg, about 1 × 10 ⁹ particles or less). Thus, the dosage may be, for example, about 1 × 10 ⁶ particle units (pu), about 2 × 10 ⁶ pu, about 4 × 10 ⁶ pu, about 1 × 10 ⁷ pu, about 2 × 10 ⁷ pu, about 4 × 10 ⁷ pu, about 1 × 10 ⁸ pu, about 2 × 10 ⁸ pu, about 4 × 10 ⁸ pu, about 1 × 10 ⁹ pu, about 2 × 10 ⁹ pu, about 4 × 10 ⁹ pu, about 1 × 10 ¹⁰ pu, about 2 × 10 ¹⁰ pu, about 4 × 10 ¹⁰ pu, about 1 × 10 ¹¹ pu, about 2 × 10 ¹¹ pu, about 4 × 10 ¹¹ pu, about 1 × 10 ¹² pu, about 2 × 10 ¹² pu Or a single dose of an adenoviral vector comprising about 4 × 10 ¹² pu of adenoviral vector. For example, Nabel, et. al. See the adenoviral vector of U.S. Pat. No. 8,454,972 B2 (incorporated herein by reference) and the dosage at column 29, lines 36-58. In certain embodiments herein, adenovirus is delivered in multiple doses.

In certain embodiments herein, delivery is via AAV. A therapeutically effective dosage for in vivo delivery of AAV to humans is in the range of about 20 to about 50 ml of saline containing about 1 × 10 ¹⁰ to about 1 × 10 ¹⁰ functional AAV / ml solution. Conceivable. Dosage may be adjusted to balance the therapeutic benefit with any side effects thereto. In certain embodiments herein, the AAV dose is generally about 1 × 10 ⁵ to 1 × 10 ⁵⁰ genomes of AAV, about 1 × 10 ⁸ to 1 × 10 ²⁰ genomes of AAV, about 1 × 10 ¹⁰ to about A concentration range of AAV from 1 × 10 ¹⁶ genomes, or from about 1 × 10 ¹¹ to about 1 × 10 ¹⁶ genomes. The human dosage may be about 1 × 10 ¹³ genomes of AAV. Such concentrations can be delivered in a carrier solution of about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml. Other effective dosages can be readily established by those skilled in the art through routine testing to generate dose response curves. For example, Hajjar, et al. U.S. Pat. No. 8,404,658 B2, column 27, lines 45-60.

  In certain embodiments herein, delivery is via a plasmid. For such plasmid compositions, the dosage must be a sufficient amount of plasmid to elicit a response. For example, a suitable amount of plasmid DNA in a plasmid composition can be from about 0.1 to about 2 mg, or from about 1 μg to about 10 μg.

  The doses herein are based on an average 70 kg individual. The frequency of administration is within the discretion of a medical or veterinary practitioner (eg, a physician, veterinarian) or a scientist skilled in the art.

  Viral vectors can be injected into the target tissue. Due to cell type specific genomic modification, the expression of Cas9 can be driven by a cell type specific promoter. For example, liver-specific expression can use the albumin promoter and nerve cell-specific expression can use the synapsin I promoter.

  RNA delivery: CRISPR enzymes, such as Cas9, and / or any of the present RNAs, such as guide RNAs, can also be delivered in the form of RNA. Cas9 mRNA can be generated using in vitro transcription. For example, Cas9 mRNA is synthesized from a β globin-poly A tail (a series of 120 or more adenines) using a PCR cassette containing the following elements: T7_promoter-Kozak sequence (GCCCACC) -Cas9-3′UTR be able to. This cassette can be used for transcription by T7 polymerase. Guide RNA can also be transcribed using in vitro transcription from a cassette containing the T7_promoter-GG-guide RNA sequence.

  To enhance expression and reduce toxicity, the CRISPR enzyme and / or guide RNA can be modified using pseudo-U or 5-methyl-C.

  CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes.

  For example, Su X, Flicke J, Kavanag DG, Irvine DJ (“In vitro and in vivo mRNA delivery using the pH-responsive polymer nanoparticles with lipid envelopes” Mol Pharm. 2011 Jun 6; 8 (3): 774-87.doi: 10.1021 / mp100390w. Electronic Publishing Apr. 1, 2011) is a poly (β-amino ester) (PBAE) core is phosphorous. A nanoparticle having a coated biodegradable core-shell structure encased in a lipid bilayer shell is described. These were developed for in vivo mRNA delivery. While the pH-responsive PBAE component was selected to promote endosomal disruption, the lipid surface layer was selected to minimize the toxicity of the polycation core. This is therefore preferred for delivery of the RNA of the invention.

  Furthermore, Michael SD Kormann et al. ("Expression of therapeutic proteins after delivery of chemically modified mRNA in mice": Nature Biotechnology, Volume 11: 1, 20: 11:29, pp. 20: 29: 1, 29: 1 Jan. 9) describes the delivery of RNA using a lipid envelope, which is also preferred in the present invention.

  mRNA delivery methods are currently particularly promising for liver delivery.

  CRISPR enzyme mRNA and guide RNA may also be delivered separately. The CRISPR enzyme mRNA can be delivered prior to the guide RNA to provide time for the CRISPR enzyme to be expressed. The CRISPR enzyme mRNA can be administered 1 to 12 hours (preferably about 2 to 6 hours) before guide RNA administration.

  Alternatively, the CRISPR enzyme mRNA and guide RNA can be administered together. Advantageously, a second booster dose of guide RNA can be administered 1-12 hours (preferably about 2-6 hours) after the initial administration of CRISPR enzyme mRNA + guide RNA.

  Further administration of CRISPR enzyme mRNA and / or guide RNA may be useful to achieve the most efficient level of genomic modification.

  In order to minimize toxicity and off-target effects, it is important to control the concentration of CRISPR enzyme mRNA and guide RNA delivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing various concentrations in a cell or animal model and analyzing the extent of modification at potential off-target genomic loci using deep sequencing. . For example, for a guide sequence that targets 5′-GAGTCCGAGCCAGAAGAAGAAA-3 ′ of the EMX1 gene of the human genome, using deep sequencing, the following two off-target loci: 1: 5′-GAGTCCTAGCAGGGAGAAGAA-3 ′ and 2 : The level of modification in 5′-GAGCTCAGAGCAGAAGAAAAA-3 ′ can be evaluated. The concentration that yields the highest on-target modification level while minimizing the off-target modification level should be selected for in vivo delivery.

  Alternatively, in order to minimize toxicity levels and off-target effects, a pair of guide RNAs targeting CRISPR enzyme nickase mRNA (eg, S. pyogenes Cas9 with D10A mutation) to the site of interest Can be delivered. The two guide RNAs must be separated as follows: Red (single underline) and blue (double underline) guide sequences, respectively (these examples are based on the PAM requirements of Streptococcus pyogenes Cas9).

  By further exploring this system, Applicants have gained evidence of 5 ′ overhangs (eg, Ran et al., Cell. 2013 Sep 12; 154 (6): 1380-9 and August 28, 2013). (See US Provisional Patent Application No. 61 / 871,301 filed on the same day). Applicants have further identified parameters associated with efficient cleavage by the Cas9 nickase mutant when combined with two guide RNAs, including but not limited to 5 'overhangs. Length is included. In embodiments of the invention, the 5 'overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In an embodiment of the invention, the 5 'overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs or 1-34 base pairs. In another preferred method of the invention, the first guide sequence directs cleavage of one strand of the DNA duplex in the vicinity of the first target sequence and the second guide sequence is the second target sequence. Directing cleavage of the other strand in the vicinity results in blunt ends or 3 ′ overhangs. In embodiments of the invention the 3 'overhang is at most 150, 100 or 25 base pairs or at least 15, 10 or 1 base pairs. In a preferred embodiment, the 3 'overhang is 1-100 base pairs.

  Aspects of the invention are mediated by reducing the expression of the gene product, or by further introducing a template polynucleotide into the DNA molecule encoding the gene product, or by annealing and ligating two 5 ′ overhangs. It relates to the precise excision of a sequence, or to alter the activity or function of a gene product, or to increase the expression of a gene product. In certain embodiments of the invention, the gene product is a protein.

  Only sgRNA pairs that produced 5 'overhangs with less than 8 bp overlap between guide sequences (offset greater than -8 bp) were able to mediate detectable indel formation. Importantly, each guide used in these assays can efficiently induce indels when paired with wild-type Cas9, and relative to the guide pair in predicting dual nicking activity. It is shown that the position is the most important parameter.

  Since Cas9n and Cas9H840A nick the opposite strand of DNA, replacement of Cas9n with Cas9H840A with a given sgRNA pair should result in an overhanging inversion. For example, a pair of sgRNAs that can generate a 5 'overhang in Cas9n should in principle generate a corresponding 3' overhang instead. Thus, sgRNA pairs that produce 3 'overhangs in Cas9n can be used to generate 5' overhangs in Cas9H840A. Unexpectedly, Applicants tested Cas9H840A with a set of sgRNA pairs designed to generate both 5 'and 3' overhangs (offset range -278 to +58 bp), but indel formation Could not be observed. Further studies may be necessary to identify the design criteria necessary to combine sgRNA pairs to allow for double nicking with Cas9H840A.

  Additional delivery options for the brain include encapsulating CRISPR enzyme and guide RNA in either DNA or RNA form in liposomes, conjugated to a molecular Trojan horse, and passing through the blood brain barrier (BBB). For example. Molecular Trojan horses have been shown to be effective in delivering B-gal expression vectors to the brain of non-human primates. The same approach can be used to deliver vectors containing CRISPR enzyme and guide RNA. For example, Xia CF and Boado RJ, Pardridge WM (“Antibody-mediated targeting of siRNA via the human instining insulin inducing instintiining insultinining insultinining insulin inducing insulin inducing insulin insulative instituting in human insulin receptor.” Mol Pharm. 2009 May-Jun; 6 (3): 747-51.doi: 101021 / mp80000194) is used in culture and in vivo by a combination of receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. How small interfering RNA (siRNA) can be delivered to cells To have. The authors also observed that the binding between the targeting mAb and siRNA is stable with avidin-biotin technology, and RNAi effects at remote sites such as the brain are observed in vivo after intravenous administration of targeted siRNA. Also report that.

  Zhang Y, Schlachtzki F, Pardridge WM. (“Global non-virtual gene transfer to the private brain following intravenous administration.” Mol Ther. 2003 Jan; 7 (1): 11-8.) That encodes a reporter, eg, a luciferase that encodes a human luciferase. It describes how it was encapsulated inside an “artificial virus” containing 85 nm pegylated immunoliposomes targeted to the rhesus monkey brain in vivo by a monoclonal antibody (MAb) against the body (HIR). HIRMAb allows liposomes carrying foreign genes to undergo transcytosis across the blood brain barrier and endocytosis across the neuronal membrane after intravenous injection. The level of luciferase gene expression in the brain was 50 times higher in rhesus monkeys compared to rats. Extensive neuronal expression of the β-galactosidase gene in the primate brain has been demonstrated by both histochemistry and confocal microscopy. The authors have shown that this approach allows reversible adult transgenics to be realized in 24 hours. Therefore, the use of immunoliposomes is preferred. They are used in conjunction with antibodies to target specific tissue or cell surface proteins.

  Nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., “Low Lipid-like nanoparticulates for small interfering RNA delivery to endothelial cells ", Advanced Functional Materials, et al., 118, 19: 3. , Levins, C., Cortez, C., Langer, R., and Anderson, D., “Lipid-based RNA for siRNA delivery. Treatment (Lipid-based nanotherapeutics for siRNA delivery) ", Journal of Internal Medicine, 267: 9-21,2010, PMID: such as through the 20,059,641), also preferred other means of delivery or RNA. In fact, exosomes have been shown to be particularly useful in the delivery of siRNA, a system that is somewhat similar to the CRISPR system. For example, El-Andaloussi S, et al. (Exosome-mediated delivery of siRNA in vitro and in vivo), Nat Protoc. 2012 Dec; 7 (12): 2112-26.doi: 10.1038 / nprot. 2012.131, Electronic Publishing, November 15, 2012) how exosomes are promising tools for drug delivery across various biological barriers and use it for siRNA delivery in vitro and in vivo It describes what you can do. Our approach is to generate targeted exosomes by transfection of expression vectors, including exosomal proteins fused to peptide ligands. The exosome is then purified from the transfected cell supernatant and characterized, and then the exosome is loaded with siRNA.

  One aspect of target sequence manipulation also relates to epigenetic manipulation of the target sequence. This may include, for example, alteration of the methylation status of the target sequence (ie, addition or removal of methylation or methylation patterns or CpG islands), histone modifications, increased or decreased accessibility to the target sequence, or 3D folding. It can be in the chromatin state of the target sequence by promotion or reduction.

  A vector is a viral or yeast system (eg, where the nucleic acid of interest can be operably linked to a promoter and is under the control of the promoter (for expression, eg, to provide the final processed RNA) It can mean not only direct delivery of the nucleic acid into the host cell.

  The present invention also provides, in certain embodiments, an abnormality in a target sequence in a target genomic locus in a subject or non-human subject in need of treatment or inhibition of a disease state caused by the target sequence abnormality in the target genomic locus. A method for treating or inhibiting a pathological condition caused, comprising a vector system comprising one or more vectors comprising operably encoding for expression the composition discussed herein. Including altering the subject or non-human subject by manipulation of the target sequence, including providing a therapy that includes delivering a non-engineered composition, or the condition is susceptible to treatment or inhibition by manipulation of the target sequence And a method of manipulating a target sequence with a composition when the composition is expressed.

  In certain embodiments of the methods herein, the methods can include inducing expression, which can be inducing expression of a CRISPR enzyme and / or inducing expression of a guide, tracr or tracr mate sequence. . In certain embodiments of the methods herein, the organism or subject is a eukaryote or a non-human eukaryote. In certain embodiments of the methods herein, the organism or subject is a plant. In certain embodiments of the methods herein, the organism or subject is a mammal or a non-human mammal. In certain embodiments of the methods herein, the organism or subject is an algae.

  In the methods herein, the vector can be a viral vector, which is advantageously AAV, while other viral vectors discussed herein can be used. For example, baculovirus can be used for expression in insect cells. These insect cells can then be useful in the production of large quantities of additional vectors, eg, AAV vectors adapted for delivery of the present invention.

  Also contemplated is a method of delivering the present CRISPR enzyme comprising delivering to cellular mRNA encoding the CRISPR enzyme. The CRISPR enzyme is truncated, is of a defined size as described herein, is a nuclease or nickase or generic DNA binding protein, is codon optimized and contains one or more mutations And / or include chimeric CRISPR enzymes, or other options discussed herein.

  Also contemplated are methods of preparing the vectors of the present invention or the present CRISPR enzyme and for use in the present methods.

  AAV viral vectors are preferred. Thus, in a further aspect, a method of preparing an AAV viral vector, comprising transfecting a plasmid containing or consisting essentially of a nucleic acid molecule encoding AAV into AAV infected cells, and AAV replication and A method is provided that includes providing an AAVrep and / or cap essential for packaging. In this regard, the CRISPR enzyme is truncated, contains less than 1000 amino acids or less than 4000 amino acids, is a nuclease or nickase, is codon optimized, contains one or more mutations, and / or It is recognized that it contains the chimeric CRISPR enzyme discussed in the literature. In some embodiments, cells are transfected with a helper plasmid or helper virus to provide AAV rep and / or cap essential for AAV replication and packaging. In some embodiments, the helper virus is a poxvirus, adenovirus, herpes virus or baculovirus. In some embodiments, the poxvirus is cowpox virus. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an insect cell and the helper virus is a baculovirus.

  The present invention, in certain embodiments, further includes a modified CRISPR enzyme. Differences from the wild type CRISPR enzyme may include: the modified CRISPR enzyme has been truncated compared to the wild type CRISPR enzyme, or the CRISPR enzyme has a defined size, eg, at least 500 amino acids, at least 800- 899 amino acids, at least 900-999 amino acids, at least 1000-1099 amino acids, at least 1100-1199 amino acids, at least 1200-1299 amino acids, at least 1300-1399 amino acids, at least 1400-1499 amino acids, at least 1500-1599 amino acids, at least 1600-1699 amino acids Or of at least 2000 amino acids; and / or the CRISPR enzyme directs cleavage of both strands in the localization of the target sequence. A lyase, or a CRISPR enzyme is a nickase that directs cleavage of one strand in the localization of the target sequence, or a CRISPR enzyme is a catalytically ineffective mutant that functions as a DNA binding protein; and The CRISPR enzyme is codon optimized or codon optimized for expression in a eukaryotic cell and / or the CRISPR enzyme contains one or more mutations and / or CRISPR The enzyme comprises a chimeric CRISPR enzyme, and / or the CRISPR enzyme has one or more other attributes discussed herein. Thus, in certain embodiments, a CRISPR enzyme may contain one or more mutations in a catalytic residue, such as spCa9D10A, E762A, H840A, N854A, N863A or D986A, or other Cas enzymes with respect to positional numbering of SpCas9 (herein). And / or have one or more mutations in the CRISPR enzyme RuvCI, RuvCII, RuvCIII, HNH or other domains described herein, and / or Alternatively, the CRISPR enzyme has one or more mutations in the catalytic domain, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence is sequence specific binding of the CRISPR complex to the target sequence. The enzyme is a functional domain Further comprising a. Functional domains include but are not limited to transcriptional activators, transcriptional repressors, recombinases, transposases, histone remodelers, DNA methyltransferases, cryptochromes, light-inducible / regulatory domains or chemically-inducible / Regulatory domains. The CRISPR enzyme may have a functional domain that in some embodiments is a transcriptional activator domain, eg, VP64. In some embodiments, the transcriptional repressor domain is KRAB. In some embodiments, the transcriptional repressor domain is a SID, or a concatemer of SIDs (ie, SID4X). In some embodiments, an epigenetic modifying enzyme is provided.

  In a preferred embodiment, the CRISPR enzyme is a Cas enzyme, such as a Cas9 ortholog.

  Aspects of the invention also include identifying novel orthologs of the CRISPR enzyme. A method for identifying a novel ortholog of a CRISPR enzyme can include identifying a tracr sequence in the genome of interest. The identification of the tracr sequence may relate to the following steps: a search for a direct repeat or tracr mate sequence in a database to identify the CRISPR region containing the CRISPR enzyme, eg, FIG. Search for homologous sequences in the CRISPR region flanking the CRISPR enzyme in both sense and antisense orientation. Search for transcription terminators and secondary structures. Identification of any sequence that is not a direct repeat or tracr mate sequence but has more than 50% identity with a direct repeat or tracr mate sequence as a potential tracr sequence. Extraction of potential tracr sequences and analysis of associated transcription terminator sequences.

  The present invention, in certain embodiments, encompasses the use of the compositions or CRISPR enzymes described herein in medicine. The present invention, in certain embodiments, further includes a composition of the invention or a CRISPR enzyme used in the methods of the invention. The present invention also includes, in certain embodiments, preferably the use of a composition or CRISPR enzyme of the invention in ex vivo gene or genome editing, or preferably in an ex vivo gene or genome editing method. Accordingly, the present invention also includes, in certain embodiments, the use of a composition of the present invention or CRISPR enzyme in the manufacture of a medicament for ex vivo gene or genome editing or used in the methods discussed herein. To do.

  Furthermore, the present invention provides that, in certain embodiments, the target sequence is flanked by a PAM suitable for the CRISPR enzyme, typically Cas, especially Cas9, or the target sequence is followed by its PAM at its 3 ′ end. Includes an inventive composition or a CRISPR enzyme. This PAM sequence is specific for each Cas9, but can be readily determined by the methods described herein.

  For example, a suitable PAM is 5'-NRG or 5'-NNGRR for the SpCas9 or SaCas9 enzyme (or derived enzyme), respectively. It is recognized herein that reference is made to Staphylococcus aureus, preferably by way of example, Staphylococcus aureus subspecies Aureus.

  Accordingly, the purpose of the present invention is not to include in the present invention any known product, method of making the product, or method of using the product, so that Applicants All rights reserved and disclaimer of any already known products, methods of making, or methods of use are disclosed herein. The present invention relates to the production of any product, method or product that does not meet the description and enablement requirements of USPTO (US Patent Act 112, first paragraph) or EPO (European Patent Convention Article 83). The method of using the product is not intended to fall within the scope of the present invention so that Applicants reserve the right to use any previously described product, method of making the product, or production thereof. It is further noted that an abandonment of how to use an object is disclosed herein.

  In this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like have the meanings ascribed to U.S. patent law. For example, they may mean “includes”, “included”, “including”, etc .; “consisting essentially of” and Terms such as “consists essentially of” have the meanings ascribed to U.S. patent law, for example, they allow elements not explicitly described but are found in the prior art. Elements that are issued or affect the basic or novel features of the present invention It is noted that to eliminate.

  These and other embodiments are disclosed or will be apparent from and encompassed by the following detailed description.

  The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Schematic representation of Cas9 nuclease guided by RNA. Cas9 nuclease from Streptococcus pyogenes is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20 nt guide sequence and a scaffold. The guide sequence base pairs with the DNA target immediately upstream of the required 5′-NGG protospacer adjacent motif (PAM), and Cas9 is a double strand break (DSB) approximately 3 bp upstream of PAM (indicated by triangles). Mediate. 2 shows the exemplary CRISPR system and possible mechanism of action (A), exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating nuclear localization and CRISPR activity (BF). 2 shows the exemplary CRISPR system and possible mechanism of action (A), exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating nuclear localization and CRISPR activity (BF). 2 shows the exemplary CRISPR system and possible mechanism of action (A), exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating nuclear localization and CRISPR activity (BF). 2 shows the exemplary CRISPR system and possible mechanism of action (A), exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating nuclear localization and CRISPR activity (BF). 2 shows the exemplary CRISPR system and possible mechanism of action (A), exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating nuclear localization and CRISPR activity (BF). 2 shows the exemplary CRISPR system and possible mechanism of action (A), exemplary adaptations for expression in eukaryotic cells, and the results of tests evaluating nuclear localization and CRISPR activity (BF). 1 shows a schematic representation of an AAV in vivo delivery plasmid utilizing an inverted terminal repeat (ITR) sequence and a guide chimeric RNA to favorably support delivery by AAV or AAV related systems. A circular representation of a phylogenetic analysis revealing five families of Cas9 including three groups of large Cas9 (about 1400 amino acids) and two groups of small Cas9 (about 1100 amino acids). A circular representation of a phylogenetic analysis revealing five families of Cas9 including three groups of large Cas9 (about 1400 amino acids) and two groups of small Cas9 (about 1100 amino acids). A circular representation of a phylogenetic analysis revealing five families of Cas9 including three groups of large Cas9 (about 1400 amino acids) and two groups of small Cas9 (about 1100 amino acids). A circular representation of a phylogenetic analysis revealing five families of Cas9 including three groups of large Cas9 (about 1400 amino acids) and two groups of small Cas9 (about 1100 amino acids). A linear representation of a phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). A linear representation of a phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). A linear representation of a phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). A linear representation of a phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). A linear representation of a phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). A linear representation of a phylogenetic analysis revealing five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids). The graph showing the length distribution of Cas9 ortholog is shown. The display of the PAM sequence logo of Cas9 ortholog as the reverse complement is shown. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. tracr mate: shows 18 chimeric RNA structures that conserve the sequence and secondary structure of the tracr sequence duplex while shortening the region that base pairs and fuses two RNA elements through an artificial loop. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. FIG. 9 shows a list of human codon optimized Cas9 sequences for pairing with the chimeric guide RNA provided in FIGS. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. The sequence at which the mutation point is located within the SpCas9 gene is shown. Figure 2 shows type II CRISPR loci in different organisms. Guide RNA sequences corresponding to CRISPR loci in different organisms are shown. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. 2 shows a table listing Cas9 orthologs and their corresponding PAM sequences. FIG. 5 shows that the PAM for Staphylococcus aureus subspecies Aureus Cas9 is NNGRR. Figure 2 shows single and multiple vector designs for SaCas9. Shown are the vector design and gel images for the Cas9 ortholog and each sgRNA used to cleave the two candidate targets present in the pUC19 base library. In vitro cleavage by SpCas9, St3Cas9, Sp_St3 chimera and St3_Sp chimera is shown. The PAM for St3Cas9 and St3_Sp chimeric Cas9 is NGG. An image of a CRISPR web server is shown. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A multiple sequence alignment for 12 Cas9 orthologs is shown. Two catalytic residues are highlighted. The first highlighted residue is a catalytic Asp residue in the RuvCI domain, and the second highlighted residue is a catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert Cas9 to nickase. Mutation of both residues converts Cas9 into a catalytically ineffective mutant useful for global DNA binding. A Western blot showing that Cas9 ortholog is expressed in HEK293FT cells is shown; DNA plasmid encoding Cas9 ortholog was transfected into HEK293FT cells and cell lysates were collected for Western blot. Cas9 ortholog # 8 is not transfected with DNA. Shows in vitro cleavage of candidate targets on pUC19 plasmid by 10 Cas9 orthologs. A consensus PAM is predicted by the sequence logo (FIG. 7), and candidate targets on pUC19 are selected based on it. Seven of the nine new Cas9 orthologs tested successfully cleaved at least one pUC19 target. SpCas9 can also cleave NGA in vitro. Shown is a representative Surveyor gel showing genomic cleavage by SaCas9. The genome cleavage efficiency of the PAM sequence (all targets) is shown. PAM sequence (cleavage target) shows genome cleavage efficiency. The genome cleavage efficiency of PAM sequences (discarding all targets, low efficiency and orphan targets) is shown. The genomic cleavage efficiency of the PAM sequence (cleavage target, low efficiency and orphan target discarded) is shown. The sequence logo for the functional cleavage spacer and PAM (new endogenous genomic test showing that T is not required) is shown.

  The drawings in this specification are for illustrative purposes only and are not necessarily drawn to scale.

  The present invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression including sequence targeting for the CRISPR-Cas system and its components, eg, genomic perturbation or gene editing. In an advantageous embodiment, the CRISPR enzyme is a Cas enzyme, such as a Cas9 ortholog.

  The present invention uses nucleic acids to bind target DNA sequences. This is advantageous because nucleic acids are much easier and cheaper to produce than, for example, peptides and can vary in specificity depending on the length of the stretch for which homology is desired. For example, a complicated three-dimensional arrangement of a plurality of fingers is not necessary.

  The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. These refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic acid-like structures with a synthetic backbone, see, eg, Eckstein, 1991; Baserga et al. Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. Nucleotide structure modifications, if present, can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

  In embodiments of the invention, the terms “chimeric RNA”, “chimeric guide RNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are used interchangeably and refer to guide sequences, tracr sequences and tracrs. Refers to a polynucleotide sequence containing a mate sequence. The term “guide sequence” refers to an approximately 20 bp sequence within a guide RNA that defines a target site, and can be used interchangeably with the terms “guide” or “spacer”. The term “tracr mate sequence” can also be used interchangeably with the term “direct repeat”.

  The term “wild type” as used herein is a term of the art as understood by one of ordinary skill in the art and is a naturally occurring organism, strain, gene or as distinguished from mutant or variant forms. Means a typical form of a feature.

  As used herein, the term “variant” should be taken to mean a presentation of quality having a pattern that deviates from that occurring in the natural state.

  The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate artificial involvement. The term refers to a nucleic acid molecule or polypeptide that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which they are naturally associated or found in the natural state. Means.

  “Complementarity” refers to the ability of a nucleic acid to form a hydrogen bond with another nucleic acid sequence, either by classical Watson-Crick base pairing or by other non-classical types. The percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5, 6, 7, 10 out of 10). 8, 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementarity). “Completely complementary” means that all consecutive residues of a nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, “substantially complementary” is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% for a region of 25, 30, 35, 40, 45, 50, or more nucleotides , 98%, 99%, or 100%, or two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization are conditions under which a nucleic acid having complementarity to a target sequence hybridizes predominantly with a target sequence and does not substantially hybridize with a non-target sequence. Point to. Stringent conditions are generally sequence-dependent and will vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions, Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay" Elsevier, N .; Y. Is described in detail. Where reference is made to a polynucleotide sequence, complementary or partially complementary sequences are also envisioned. They are preferably capable of hybridizing to the reference sequence under highly stringent conditions. Generally, in order to maximize the rate of hybridization, relatively low stringency hybridization conditions are selected: about 20-25 ° C. below the thermal melting point (T _m ). T _m is the temperature at which 50% of a particular target sequence hybridizes with a fully complementary probe in a solution of defined ionic strength and pH. In general, highly stringent wash conditions are selected to determine at least about 85% nucleotide complementarity of the hybridized sequences by about 5-15 ° C. below the T _m . To determine at least about 70% nucleotide complementarity of the hybridized sequences, moderately stringent wash conditions are selected that are about 15-30 ° C. below the T _m . Highly permissive (very low stringency) wash conditions may be as low as 50 ° C. below the T _m , allowing a high degree of mismatch between the hybridized sequences. Those skilled in the art will appreciate that other physical and chemical parameters in the hybridization and wash steps will affect the outcome of the detectable hybridization signal due to the specific level of homology between the target and probe sequences. Will also recognize that it may change. Preferred highly stringent conditions are: 50% formamide, 5 × SSC, and 1% SDS, 42 ° C. incubation, or 5 × SSC and 1% SDS, 65 ° C. incubation, 0.2 × SSC and Wash with 0.1% SDS, 65 ° C.

  “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen bonding, or in any other sequence specific manner. The complex may comprise two strands forming a double stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a wider range of processes, such as initiation of PCR or enzymatic cleavage of a polynucleotide. A sequence that can hybridize to a given sequence is referred to as the “complementary strand” of the given sequence.

  As used herein, the term “genomic locus” or “locus” (locus in plural form) refers to a specific location of a gene or DNA sequence on a chromosome. It is. “Gene” refers to a stretch of DNA or RNA encoding a polypeptide, or an RNA strand that plays a functional role in an organism and thus is a molecular unit of inheritance in the organism. For the purposes of the present invention, a gene can be considered to include a region that regulates the production of a gene product (whether or not such regulatory sequences are flanked by coding and / or transcribed sequences). Thus, genes are not necessarily limited, but include promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and in-sequence ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment Site and locus control regions are included.

  As used herein, “genomic locus expression” or “gene expression” is the process by which information from a gene is used in the synthesis of a functional gene product. The product of gene expression is often a protein, but for non-protein-encoding genes such as rRNA genes or tRNA genes, the product is functional RNA. The gene expression process is used by all known life-eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses to generate functional products for survival. As used herein, “expression” of a gene or nucleic acid includes not only cellular gene expression, but also transcription and translation of the nucleic acid in cloning systems and in any other context. As used herein, “expression” also refers to the process by which a polynucleotide is transcribed from a DNA template (eg, to an mRNA or other RNA transcript) and / or the transcribed mRNA is subsequently a peptide, polypeptide. Or a process that is translated into protein. Transcripts and encoded polypeptides may be collectively referred to as “gene products”. If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched and it can contain modified amino acids, which can be interrupted by non-amino acids. The term also encompasses amino acid polymers that have undergone modifications such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The term “amino acid” as used herein includes natural and / or non-natural or synthetic amino acids, including both glycine and the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

  As used herein, the term “domain” or “protein domain” refers to a portion of a protein sequence that can exist and function independently of the rest of the protein chain.

  As described in aspects of the invention, sequence identity is related to sequence homology. Homology comparisons may be made by eye, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent (%) homology between two or more sequences and can also calculate sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homology can be generated by any of a number of computer programs known in the art, such as BLAST or FASTA. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al., 1999 supra-Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and GENEWORKS suite comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 supra, pages 7-58 to 7-60). However, it is preferred to use the GCG Bestfit program.

  % Sequence homology can be calculated over contiguous sequences, ie, one sequence is aligned with the other sequence, and each amino acid or nucleotide of one sequence is one at a time with the corresponding amino acid or nucleotide of the other sequence Are directly compared. This is called a “no gap” alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

  This is a very simple and consistent method, but for example, in sequence pairs that are identical in nature, a single insertion or deletion causes a shift in the alignment of subsequent amino acid residues, and thus% homology when performing a global alignment. Cannot be taken into account. As a result, many sequence comparison methods are designed to create optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology or identity score. . This is accomplished by inserting “gaps” in the sequence alignment to try to maximize local homology or identity.

  However, in these more complex methods, each gap appearing in the alignment is assigned a “gap penalty”, so for the same number of identical amino acids, as little gap as possible—a higher association between the two compared sequences. A sequence alignment with-reflecting sex can achieve a higher score than one with many gaps. “Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties can of course produce optimized alignments with fewer gaps. It is possible to change the gap penalty in many alignment programs. However, it is preferred to use the default values when using software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

Therefore, the calculation of maximum% homology first requires creating an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984 Nuc. Acids Research 12 p387). Examples of other software that can perform sequence comparisons include, but are not limited to, BLAST package (Ausubel et al., See ^{1999 Short Protocols in Molecular Biology, 4} th Ed.-Chapter 18), FASTA (Altschul et al , 1990 J. Mol. Biol. 403-410) and GENEWORKS suite comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, it is preferable to use the GCG Bestfit program for some applications. A new tool called BLAST 2 Sequences is also available for protein and nucleotide sequence comparison (FEMS Microbiol Lett. 1999 174 (2): 247-50; FEMS Microbiol Lett. 1999 177 (1): 187-8 and the United States (See National Center for Biotechnology Information website at the National Institutes of Health website).

  Although the final% homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns a score to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix-BLAST program suite default matrix. The GCG Wisconsin program generally uses publicly available default values or a custom symbol comparison table if available (see user manual for further details). Depending on the application, it is preferable to use the public default values of the GCG package, or in the case of other software, a default matrix such as BLOSUM62.

  Alternatively, the percentage homology is calculated based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73 (1), 237-244) using the multiple alignment function of DNASIS ™ (Hitachi Software). can do. Once the software has created an optimal alignment, it is possible to calculate% homology, preferably% sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

  The sequence may also have deletions, insertions or substitutions of amino acid residues that cause silent changes resulting in functionally equivalent material. Planned amino acid substitutions may be made based on similarities in amino acid properties (such as residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic nature), thus making amino acids functional groups It is useful to combine them together. Amino acids may be grouped together based solely on their side chain properties. However, it is more useful to include mutation data as well. The amino acid set thus obtained appears to be conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical" analysis of residue conservation) "Comput.Appl.Biosci.9: 745-756) (Taylor WR (1986)" The classification of amino acid conservation "J. Thel. -218). Conservative substitutions can be made, for example, according to the table below that describes the amino acid classification according to the generally accepted Venn diagram.

  Embodiments of the present invention use the possible homologous substitutions (substitution and substitution) herein to mean interchange of existing amino acid residues or nucleotides with alternative residues or nucleotides. In other words, in the case of amino acids, sequences (both polynucleotides or polypeptides) that may contain substitutions of the same species, such as basic, acidic, polar, etc. Non-homologous substitution, ie, substitution from one class to another, ornithine (hereinafter referred to as Z), ornithine diaminobutyrate (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O) ), Substitutions involving the incorporation of unnatural amino acids such as pyridylalanine, thienylalanine, naphthylalanine and phenylglycine can also occur.

  A variant amino acid sequence can be inserted between any two amino acid residues of a sequence containing an alkyl group such as a methyl, ethyl or propyl group in addition to an amino acid spacer such as a glycine or β-alanine residue. A spacer group. Those of ordinary skill in the art are well aware of alternative forms of change that involve the presence of one or more amino acid residues in the peptoid form. To avoid misunderstanding, “peptoid form” is used to refer to variant amino acid residues in which the α-carbon substituent is not on the α-carbon, but rather on the nitrogen atom of the residue. Methods for preparing peptides in the peptoid form are known in the art (eg, Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134).

  The practice of the present invention, unless otherwise stated, employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the artisan. Use. Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) See ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CURRENT (RI Freshney, ed. (1987)).

  In one aspect, the present invention provides vectors for use in engineering and optimization of the CRISPR-Cas system.

  As used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. A vector is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted, resulting in replication of the inserted segment. In general, vectors are capable of replication when associated with appropriate control elements. In general, the term “vector” refers to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules that include one or more free ends and do not include free ends (eg, circular); Nucleic acid molecules comprising DNA, RNA, or both; and other polynucleotide variants known in the art. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which a DNA or RNA sequence from a virus for packaging into a virus (eg, retrovirus, replication defective retrovirus, adenovirus, replication defective adenovirus, and adeno-associated virus). Is present in the vector. Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Certain vectors have the ability to self-replicate in the host cell into which it is introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and thus replicate with the host genome. In addition, certain vectors have the ability to direct expression of genes that are operably linked. Such vectors are referred to herein as “expression vectors”. Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Further discussion of vectors is provided herein.

  A recombinant expression vector may comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which includes one or more operably linked nucleic acid sequences to be expressed by the recombinant expression vector. It is meant to include regulatory elements, which can be selected based on the host cell used for expression. “Operatively linked” within a recombinant expression vector means that the nucleotide sequence of interest is introduced into a regulatory element and that nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell). Is intended to mean bound in a manner that allows expression in a host cell. Regarding recombination and cloning methods, US Patent Application No. 10 / 815,730 published as US Patent Application Publication No. 2004-0171156 A1 on September 2, 2004 (the contents of which are generally referred to). Incorporated herein by reference).

  Aspects and embodiments of the invention relate to bicistronic vectors for chimeric RNAs and Cas9 or Cas9 orthologs. In some embodiments, Cas9 is driven by the CBh promoter. In some embodiments, the chimeric RNA is driven by a U6 promoter. Preferably, CBh and U6 are used together in the sense that Cas9 is driven by the CBh promoter and the chimeric RNA is driven by the U6 promoter. In some embodiments, the chimeric guide RNA is a 20 bp guide sequence that binds to a trcr sequence truncated from the first “U” of the lower strand to the end of the transcript, truncated at the various positions indicated. (N). The guide and tracr sequences are preferably separated by a tracr mate sequence. A preferred example of a tracr mate sequence is GUUUUAGAGCUA. This is preferably followed by a loop sequence. The loop is preferably GAAA, but is not limited to this sequence, or actually only 4 bp long. Indeed, the preferred loop forming sequence used in the hairpin structure is 4 nucleotides long and most preferably has the sequence GAAA. However, longer or shorter loop sequences can be used so that alternative sequences can be used. The sequence preferably includes a nucleotide triplet (eg, AAA) and an additional nucleotide (eg, C or G). Examples of loop forming sequences include CAAA and AAAG. Throughout this application, chimeric RNA may also be referred to as a single guide or a synthetic guide RNA (sgRNA).

  For example, as detailed in Example 2, a chimeric guide RNA can be designed as shown in FIG. The CRISPR loci in some of these families are shown in FIG. The corresponding guide RNA sequence is shown in FIG. Applicants analyzed genomic DNA sequences within about 2 kb of the Cas9 protein and identified direct repeats ranging from 35 bp to 50 bp with intervening spacers ranging from 29 bp to 35 bp. Based on direct repeat sequences, Applicants searched for tracrRNA candidate sequences having the following criteria: outside the crRNA array but containing a high degree of homology with the direct repeat (direct repeat: tracrRNA bases) Required for pairing; custom computational analysis), contains a Rho-independent transcription termination signal outside the coding region of the protein component, approximately 60 bp to 120 bp downstream from the region of homology to the direct repeat, and direct Co-folding with repeats to form a duplex, followed by two or more hairpin structures at the distal end of the tracrRNA sequence. Based on these predictive criteria, Applicants selected 18 Cas9 proteins and their uniquely related direct repeats and an initial set of tracrRNAs distributed across all five Cas9 families. Applicants have conserved the sequence and secondary structure of the natural direct repeat: tracrRNA duplex, while shortening the region that base pairs and fuses two RNA elements through an artificial loop. Further sets were generated (FIGS. 6A-J).

  The term “regulatory element” is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of nucleotide sequences in many types of host cells and those that direct expression of nucleotide sequences only in specific host cells (eg, tissue-specific regulatory sequences). Tissue-specific promoters primarily express expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, lymphocytes). Can be directed. Regulatory elements can also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific at the same time. Also good. In some embodiments, the vector has one or more pol III promoters (eg, 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (eg, 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (eg, 1, 2, 3, 4, 5, or more pol I promoters), or Includes combinations. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [eg Boshart et al, Cell, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. And enhancer elements such as WPRE; CMV enhancer; R-U5 ′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8 (1), p. 466-472, 1988); SV40 enhancer; An intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78 (3), p. 1527-31, 1981) is also included in the term “regulatory element”. Is done. It will be appreciated by those skilled in the art that the design of an expression vector can depend on factors such as the choice of host cell to transform, the desired level of expression, and the like. A vector is introduced into a host cell, whereby a transcript, protein, or peptide encoded by a nucleic acid as described herein, eg, a fusion protein or peptide (eg, clustered equidistant short batch repeat (CRISPR)) Transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.) can be produced. For regulatory sequences, reference is made to US patent application Ser. No. 10 / 491,026, the contents of which are hereby incorporated by reference in their entirety. Regarding promoters, reference is made to WO 2011/028929 and US patent application Ser. No. 12 / 511,940, the contents of which are hereby incorporated by reference in their entirety.

  Vectors can be designed for expression of CRISPR transcripts (eg, nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, the CRISPR transcript can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells include Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

  Vectors can be introduced into and proliferated in prokaryotes or prokaryotic cells. In some embodiments, prokaryotes are used to amplify a copy of the vector to be introduced into a eukaryotic cell, or used as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g., Amplify plasmid as part of viral vector packaging system). In some embodiments, prokaryotes are used to amplify a copy of the vector and express one or more nucleic acids, e.g., one or more protein resources for delivery to a host cell or host organism. provide. Expression of proteins in prokaryotes is most often performed in Escherichia coli with vectors containing constitutive or inducible promoters that direct expression of either fused or unfused proteins. . A fusion vector adds a number of amino acids to the protein encoded thereby, eg, to the amino terminus of a recombinant protein. Such a fusion vector serves as a ligand in one or more purposes, eg, (i) increased expression of the recombinant protein; (ii) increased solubility of the recombinant protein; and (iii) affinity purification. Can assist in the purification of recombinant proteins by. In fusion expression vectors, proteolytic cleavage sites are often introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia. Each of which fuses glutathione S-transferase (GST), maltose E binding protein, or protein A to the target recombinant protein.

  Examples of suitable inducible non-fused E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS: IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

  In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell). : 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), And picZ (InVitrogen Corp, San Diego, Calif.). .

  In some embodiments, the vector uses baculovirus expression vectors to drive protein expression in insect cells. Examples of baculovirus vectors that can be used for protein expression in cultured insect cells (eg, SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series. (Lucklow and Summers, 1989. Virology 170: 31-39).

  In some embodiments, the vector can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, expression vector control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus type 2, cytomegalovirus, simian virus 40, and others disclosed herein and those known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, eg, Chapters 16 and 17 of Sambrook, et al. , MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .; Y. 1989.

  In some embodiments, the recombinant mammalian expression vector can preferentially direct expression of the nucleic acid in a particular cell type (eg, use a tissue-specific regulatory element to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue specific promoters include albumin promoters (liver specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid system specific promoters (Callame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular the promoter of the T cell receptor (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulin (Baneiji, et al., 1983. Cell 33). : 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoters; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas specific promoter (Edrund, et al., 1985. Science 230: 912-916), and mammary gland specific promoters (eg, milk A clean promoter; U.S. Pat. No. 4,873,316 and EP 264,166). Also included are developmental control promoters, such as the murine hox promoter (Kessel and Gross, 1990. Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With respect to these prokaryotic and eukaryotic vectors, reference is made to US Pat. No. 6,750,059, the contents of which are hereby incorporated by reference in their entirety. Other embodiments of the invention may relate to the use of viral vectors, in which US patent application Ser. No. 13 / 092,085, the contents of which are incorporated herein by reference in their entirety. Referenced. Tissue specific regulatory elements are known in the art and reference is made in this regard to US Pat. No. 7,776,321, the contents of which are hereby incorporated by reference in their entirety.

  In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system so as to drive expression of one or more elements of the CRISPR system. In general, CRISPR (clustered equally spaced short repeats) is also known as SPIDR (Spacer Interspersed Direct Repeat), which is usually a DNA locus that is specific for a particular bacterial species. Make up a family. The CRISPR locus is a distinct class of scattered short sequence repeats (SSR) recognized in E. coli (Ishino et al., J. Bacteriol., 169: 5429-5433 [1987]; And Nakata et al., J. Bacteriol., 171: 3553-3556 [1989]) and related genes. Similar sporadic SSRs have been identified in Haloferax meditelanei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis et. , Mol. Microbiol., 10: 1057-1065 [1993]; Hoe et al., Emerg. Infect.Dis., 5: 254-263 [1999]; 30 [1996]; and Mojica et al., Mol. Microbiol., 17: 85-93 [1995]. Reference). CRISPR loci typically differ in repeat structure from other SSRs, which are referred to as short chain equally spaced repeats (SRSR) (Janssen et al., OMICS J. Integ. Biol., 6:23). -33 [2002]; and Mojica et al., Mol. Microbiol., 36: 244-246 [2000]). In general, repeats are short elements that occur in clusters that are equally spaced by a unique intervening sequence having a substantially constant length (Mojica et al., [2000], supra). While repeat sequences are highly conserved among strains, the number of scattered repeats and the sequence of the spacer region typically vary from strain to strain (van Embden et al., J. Bacteriol., 182: 2393). 2401 [2000]). CRISPR loci have been identified in over 40 prokaryotes (see, eg, Jansen et al., Mol. Microbiol., 43: 1565-1575 [2002]; and Mojica et al., [2005]), Examples include, but are not limited to, Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocalcula, Methanobacteria (Metanobacterium) Methanobacterium), Methanococcus, Methanosarcina, Methanopyrus yrus), Pyrococcus, Picophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Streptomyces, (Aquifex), Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Lostria, Clostridium C Thermoanaerobacter (Thermoan aerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomons, Nitrosomons Geobacter, Myxococcus, Campylobacter, Warinella, Acinetobacter, Erwinia, E, Echrecia Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, T, and Treponema e It is.

  In general, the “CRISPR system” collectively refers to transcripts and other elements involved in the expression of CRISPR-related (“Cas”) genes or the direction of their activity, such as sequences encoding the Cas gene, tracr (trans Activated CRISPR) sequences (eg, tracrRNA or active partial tracrRNA), tracr mate sequences (including “direct repeats” for endogenous CRISPR systems and partial direct repeats processed by tracrRNA), guide sequences (“for endogenous CRISPR systems” Also referred to as "spacer"), or other sequences and transcripts from the CRISPR locus. In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism that includes the endogenous CRISPR system, such as Streptococcus pyogenes. In general, the CRISPR system is characterized by elements that promote the formation of a CRISPR complex in the target sequence (also referred to as a protospacer with respect to the endogenous CRISPR system). With respect to formation of a CRISPR complex, a “target sequence” refers to a sequence that is designed such that the guide sequence is complementary, and hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. The target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

  In a preferred embodiment of the invention, the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 or any closely related Cas9 from Streptococcus pyogenes hybridizes to a 20 nucleotide guide sequence, followed by a protospacer adjacent motif (PAM) sequence NGG / Double-strand breaks in target site sequences with NRG (eg, as discussed elsewhere, suitable PAMs are 5′-NRG or 5′-NNGRRR for the SpCas9 or SaCas9 enzyme (or derived enzyme), respectively) Is generated. CRISPR activity through Cas9 for site-specific DNA recognition and cleavage is defined by a guide sequence, a tracr sequence that partially hybridizes to the guide sequence, and a PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archee, Molle Cell 2010, January 15; 37 (1): 7.

  The type II CRISPR locus from Streptococcus pyogenes SF370 is composed of a cluster of four genes Cas9, Cas1, Cas2, and Csn1, and two non-coding RNA elements, tracrRNA and a short stretch of non-repetitive sequences ( Includes a characteristic array of repetitive sequences (direct repeats) between which spacers, each approximately 30 bp). In this system, a targeted DNA double-strand break (DSB) is created in four sequential steps (FIG. 2A). First, two non-coding RNAs, a pre-crRNA array and a tracrRNA are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to a direct repeat of the precrRNA, which is then processed into a mature crRNA containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM by heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of the target DNA upstream of the PAM, creating a DSB within the protospacer (FIG. 2A). FIG. 2B demonstrates the nuclear localization of codon optimized Cas9. To promote accurate transcription initiation, the RNA polymerase III-based U6 promoter was chosen to drive the expression of tracrRNA (FIG. 2C). Similarly, to express a pre-crRNA array consisting of a single spacer and two direct repeats adjacent to it (DR, also encompassed by the term “tracr mate sequence”; FIG. 2C), a U6 promoter-based construct is It has been developed. The initial spacer targets a 33 base pair (bp) target site (30 bp protospacer + 3 bp CRISPR motif (PAM) sequence satisfying the NGG recognition motif of Cas9) at the human EMX1 locus, which is a gene important for cerebral cortex development. Designed (Figure 2C).

  FIG. 16 shows that Cas9 orthologs and respective sgRNAs are used to cleave two candidate targets present in the pUC19-based library. Target 1 is followed by a randomized PAM containing 7 degenerate bases (5'-NNNNNNNN-3 '), and target 1' containing the same target sequence as target 1 is immobilized PAM (5'-TGGAGAAT- 3 ') continues. Each Cas9 ortholog sgRNA contains a guide sequence for target 1 or target 1 '. The gel image shows good cleavage by the 20 Cas9 orthologs, indicating that their sgRNA design is functional for their respective Cas9 enzyme.

  In some embodiments, the direct repeat or tracr mate sequence is downloaded from the CRISPR database, or They are identified in silico by searching for repeated motifs found in a 2 kb window of the genomic sequence flanking the type II CRISPR locus, ranging from 2.20 to 50 bp and every 3.20 to 50 bp. In some embodiments, two of these criteria may be used, for example, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used.

  In some embodiments, the candidate tracrRNA is subsequently 1. 1. Sequence homology with direct repeats (motif search in Geneious with up to 18 bp mismatch), 2. 2. the presence of a predicted Rho-independent transcription terminator in the direction of transcription, and Predicted by stable hairpin secondary structure between tracrRNA and direct repeats. In some embodiments, two of these criteria may be used, for example, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used. In some embodiments, the chimeric synthesis guide RNA (sgRNA) design incorporates a double-stranded structure of at least 8 bp between the direct repeat and the tracrRNA.

  Some aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity may depend on the availability of free Mg2 + at levels higher than those present in mammalian nuclei (see, eg, Jinek et al., 2012, Science, 337: 816) The preference for adjacent downstream NGG / NRG motifs limits the targeting ability by an average of 12 bp in the human genome.

  Typically, for an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that is hybridized to the target sequence and complexed with one or more Cas proteins) is in or near the target sequence ( For example, it then leads to cleavage of one or both strands (within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs). Without being bound by theory, the tracr sequence may be all or part of the wild type tracr sequence (eg, about or about 20, 26, 32, 45, 48, 54, 63, 67, wild type tracr sequence, At least one of the tracr sequences with all or part of the tracr mate sequence operably linked to the guide sequence, for example, 85 or more nucleotides). Part of the CRISPR complex can also be formed by hybridization along the part. In some embodiments, one or more vectors that drive the expression of one or more elements of the CRISPR system are introduced into the host cell so that expression of the elements of the CRISPR system is at one or more target sites. Directs the formation of the CRISPR complex. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence can each be operably linked to a separate regulatory element on a separate vector. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector, and one or more additional vectors that provide any component of the CRISPR system are in the first vector Not included. CRISPR-based elements that are combined in a single vector can be placed in any suitable orientation, for example, one element 5 '(upstream) or 3' (downstream) relative to a second element. ) Can be localized. The coding sequence of one element can be localized on the same or reverse strand of the coding sequence of the second element and oriented in the same or reverse orientation. In some embodiments, a single promoter is a transcript encoding the CRISPR enzyme, as well as a guide sequence, tracr mate sequence (optionally operably linked to the guide sequence) embedded within one or more intron sequences. Driven) and one or more expression of tracr sequences (eg, each in a different intron, two or more in at least one intron, or all in a single intron) To do. In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

  In some embodiments, the vector includes one or more insertion sites, eg, restriction endonuclease recognition sequences (also referred to as “cloning sites”). In some embodiments, one or more insertion sites (eg, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites). Are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence and optionally downstream of a regulatory element operably linked to the tracr mate sequence, so that a guide sequence into the insertion site. The guide sequence directs the sequence-specific binding of the CRISPR complex to the target sequence in eukaryotic cells after insertion and during expression. In some embodiments, the vector includes two or more insertion sites, each insertion site localized between two tracr mate sequences to allow insertion of a guide sequence at each site. Yes. In such an arrangement, the two or more guide sequences can include two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof. If multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity against multiple different corresponding target sequences in the cell. For example, a single vector can include about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, providing about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide sequence-containing vectors, Can be delivered to cells.

  In some embodiments, the vector comprises a regulatory element operably linked to a CRISPR enzyme, eg, an enzyme coding sequence that encodes a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2 , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx14, Csx14, Csx14 , Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, eg, Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands in the localization of the target sequence, eg, within the target sequence and / or within the complementary strand of the target sequence. In some embodiments, the CRISPR enzyme is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, from the first or last nucleotide of the target sequence. Directs cleavage of one or both strands within 100, 200, 500, or more base pairs. In some embodiments, the vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme so that the mutant CRISPR enzyme is one or both of the target polynucleotides containing the target sequence. Lacks the ability to cleave the chain. For example, substitution of aspartate to alanine (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes (D10A) cleaves Cas9 from a nuclease that cleaves both strands (single strand cleavage). To). Other examples of mutations that make Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A in SpCas9. As discussed herein, the corresponding positions can be conserved in other Cas9 from or derived from other bacterial species with respect to the position numbering of SpCas9, ie in the Cas9 ortholog. (FIG. 19) shows a multiple sequence alignment of 12 Cas9 orthologs, showing the conserved catalytic Asp residue in the RuvCI domain and the conserved catalytic His residue in the HNH domain. Mutation of one or the other residue to Ala can convert a Cas9 ortholog into a nickase. Mutation of both residues can convert the Cas9 ortholog into a catalytically ineffective mutant useful for global DNA binding. As a further example, mutating two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or HNH domains) to produce a mutant Cas9 ortholog that substantially lacks all DNA cleavage activity. it can. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, the CRISPR enzyme has a DNA cleavage activity of the mutant enzyme of about 25%, 10%, 5%, 1%, 0.1%, 0.01%, relative to its non-mutated form, If it is less than or less than that, it is considered substantially lacking all DNA cleavage activity.

  Engineering the aspartate to alanine substitution (D10A) in the RuvC I catalytic domain of SpCas9 to convert nucleases to nickases (eg, Saplanauskas et al., 2011, Nucleic Acis Research, 39: 9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109: E2579), nicked genomic DNA was allowed to undergo high fidelity homologous recombination repair (HDR). Applicants have confirmed by a Surveyor assay that SpCas9n does not generate indels to the EMX1 protospacer target. When EMX1 targeting chimeric crRNA (which also has a tracrRNA component) was coexpressed with SpCas9, an indel was generated at the target site, but not with coexpression with SpCas9n (n = 3). Furthermore, sequencing of 327 amplicons did not detect indels induced by SpCas9n. Select the same locus and co-transfect HEK293FT cells with a chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, and an HR template to introduce a pair of restriction sites (HindIII and NheI) near the protospacer Tested CRISPR-mediated HR. SpCas9 and SpCas9n actually catalyzed the integration of the HR template into the EMX1 locus. PCR amplification of the target region followed by restriction digestion with HindIII revealed cleavage products corresponding to the expected fragment size, and SpCas9 and SpCas9n mediated similar levels of HR efficiency. Applicants further validated HR using Sanger sequencing of genomic amplicons and demonstrated the utility of CRISPR to facilitate targeted gene insertion in the mammalian genome. Considering the target specificity of wild-type SpCas9 14 bp (12 bp from spacer and 2 bp from PAM), the availability of nickase is off-target because single-strand breaks are not substrates for the error-prone NHEJ pathway. The possibility of modification can be significantly reduced. FIGS. 10A-M provide schematic diagrams showing the location of mutations in SpCas9, and Cas9 orthologs typically share the general organization of 3-4 RuvC and HNH domains. Most RuvC domains in 5 'cleave non-complementary strands and HNH domains cleave complementary strands.

  The catalytic residues in the 5′RuvC domain include the target Cas9 and other Cas9 orthologs (S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus). (S. thermophilus) identified through homology comparison with CRISPR locus 3 and Franciscilla novicida type II CRISPR locus, and conserved Asp residue was mutated to alanine to complement Cas9 with complementary strand nicks Convert to forming enzyme. Similarly, conserved His and Asn residues in the HNH domain are mutated to alanine to convert Cas9 to a non-complementary strand nicking enzyme.

  In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon optimized, particularly for expression in a cell, eg, a eukaryotic cell. The eukaryotic cell may be from or derived from a particular organism, such as a mammal, such as, but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate. In some embodiments, a method of altering a human germline genetic identity and / or a method of altering an animal's genetic identity without any substantial medical benefit to a person or animal Methods that can cause distress and animals resulting from such methods can also be excluded.

  In general, codon optimization involves at least one codon of the native sequence (eg, about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons). Process of altering a nucleic acid sequence for improved expression in a target host cell by replacing the natural amino acid sequence while replacing the most frequently or most frequently used codons in the host cell gene Point to. Different species exhibit specific biases for certain codons of specific amino acids. Codon bias (difference in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn translates into the characteristics of the translated codon and the specific transfer RNA ( It is believed to be dependent on the availability of tRNA) molecules. The superiority of tRNA selected in cells is generally a reflection of the most frequently used codons in peptide synthesis. Thus, genes can be adjusted for optimal gene expression in a given organism based on codon optimization. The codon usage frequency table is, for example, www. kazusa. Available readily in the “Codon Usage Database” available at orjp / codon / (accessed July 9, 2002), these tables can be adapted in a number of ways. Nakamura, Y .; , Et al. “Codon usage table from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28: 292 (2000). Computer algorithms are also available for codon optimizing specific sequences for expression in specific host cells, such as Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons in the sequence encoding the CRISPR enzyme (eg, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or All codons) correspond to the most frequently used codons for a particular amino acid.

  In some embodiments, the vector is one or more nuclear localization sequences (NLS), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more It encodes a CRISPR enzyme containing more than a greater number of NLS. In some embodiments, the CRISPR enzyme has an NLS greater than or about about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more at or near the amino terminus, About or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLS at or near the carboxy terminus, or combinations thereof (eg, 1 at the amino terminus) One or more NLS and one or more NLS at the carboxy terminus). If more than one NLS is present, each is independent of the others so that a single NLS can exist in more than one copy and / or one present in more than one copy Selection can be made in combination with other NLS. In a preferred embodiment of the invention, the CRISPR enzyme comprises at most 6 NLS. In some embodiments, the NLS is such that the nearest amino acid of the NLS is about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 along the polypeptide chain from the N or C terminus. , 50, or more amino acids are considered to be near the N- or C-terminus. Non-limiting examples of NLS include the NLS of the SV40 viral large T antigen having the amino acid sequence PKKRKV; NLS from nucleoplasmin (eg, the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); C-mycNLS with; sequence NQSSNGFPMKGNGGFRGSSGPYGGGGQYFAKPRNQGGY with hRNPA1 M9NLS; sequence of IBB domain from importin alpha Mouse c-abl IV sequence SALIKKKKKKMAP; influenza virus NS1 sequences DRLRR and PKQKKRK; hepatitis virus delta antigen sequence RKLKKKIKKKL; mouse Mx1 protein sequence REKKKFLKRR; An NLS sequence derived from the human glucocorticoid sequence RKCLQAGMNLEEARKTKK.

  In general, the one or more NLSs are strong enough to drive the accumulation of a detectable amount of CRISPR enzyme in the nucleus of a eukaryotic cell. In general, the intensity of nuclear localization activity can be derived from the number of NLS in the CRISPR enzyme, the particular NLS used, or a combination of these factors. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to the CRISPR enzyme so that intracellular localization, eg, means for detecting nuclear localization (eg, nuclear specific staining, eg, DAPI) Can be visualized in combination with. Cell nuclei can also be isolated from cells and their contents can then be analyzed by any suitable process for detecting proteins, such as immunohistological analysis, Western blot, or enzyme activity assays. Accumulation in the nucleus is, for example, an assay for the effect of CRISPR complex formation (eg, an assay for DNA cleavage or mutation in the target sequence, or gene expression activity affected by CRISPR complex formation and / or CRISPR enzyme activity). Can also be measured indirectly compared to controls exposed to CRISPR enzymes that are not exposed to CRISPR enzyme or complex, or that lack one or more NLS.

  In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or about 50%, 60%, 75 when optimally aligned using a suitable alignment algorithm. %, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burrows-Wheeler Transform. Algorithms based (eg Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (available at Novocraft Technologies, www.novocraft, com), ELAND (IlluminoSop.com). org.cn), and Maq (maq. Available) and the like in Ourceforge.Net. In some embodiments, the guide arrangement is about or about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or less nucleotides in length. The ability of the guide sequence to direct sequence specific binding of the CRISPR complex to the target sequence can be assessed by any suitable assay. For example, sufficient components of the CRISPR system to form a CRISPR complex, eg, a guide sequence to be tested, can be transfected into a host cell having a corresponding target sequence, eg, by a vector encoding a component of the CRISPR sequence. And then preferential cleavage within the target sequence is assessed, for example, by the Surveyor assay described herein. Similarly, cleavage of the target polynucleotide sequence provides the target sequence in the test tube, a component of the CRISPR complex, eg, the guide sequence to be tested and a control guide sequence that is different from the test guide sequence. It can be assessed by comparing the ratio of binding or cleavage in the target sequence during the sequence reaction. Other assays are contemplated and will be recognized by those skilled in the art.

  Multiplexed nickase: Cas9 nickase can also be generated using the Cas9 optimization aspects and teachings detailed in this application. Cas9 nickase can advantageously be used in combination with a guide RNA pair to generate a DNA double-strand break with an overhang defined. When using two pairs of guide RNAs, it is possible to excise the intervening DNA fragment. When exogenous pieces of DNA are cleaved by two pairs of guide RNAs to produce an overhang compatible with genomic DNA, the exogenous DNA fragment can be ligated into the genomic DNA to replace the excised fragment. For example, it can be used to treat Huntington's disease by removing trinucleotide repeat extensions in the Huntington (HTT) gene.

  Cas9 and its chimeric guide RNA, or a combination of tracrRNA and crRNA can be delivered as either DNA or RNA. Delivery of both Cas9 and guide RNA as RNA (normally or containing base or backbone modifications) molecules can be used to reduce the amount of time that Cas9 protein lasts in the cell. This can reduce the level of off-target cleavage activity in the target cell. Because delivery of Cas9 as mRNA requires time to be translated into protein, guide RNA should be delivered hours after the delivery of Cas9 mRNA to maximize the level of guide RNA available for interaction with Cas9 protein Can be advantageous.

  In situations where the amount of guide RNA is limited, it may be desirable to introduce guide RNA in the form of a DNA expression cassette having a promoter that drives expression of Cas9 and DNA guide RNA as mRNA. Thus, the amount of guide RNA available is amplified via transcription.

  Various delivery systems can be introduced to introduce Cas9 (DNA or RNA) and guide RNA (DNA or RNA) into the host cell. These include the use of liposomes, viral vectors, electroporation, nanoparticles, nanowires (Shalek et al., Nano Letters, 2012), exosomes. Molecular Trojan liposomes (Pardridge et al., Cold Spring Harb Protoc; 2010; doi: 10.1101 / pdb.prot 5407) can be used to deliver Cas9 and guide RNA through the blood brain barrier.

  Guide RNA considerations for orthologs: In some embodiments, guide sequences are selected to reduce the degree of secondary structure within the guide RNA. In some embodiments, a guide of about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less nucleotides RNA is involved in self-complementary base pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, which is described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold developed at the Institute for Theoretic Chemistry at the University of Vienna using a centroid structure prediction algorithm (see, eg, AR Gruber et al., 2008). 106 (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62). A method for optimizing the guide RNA of the Cas9 ortholog involves destroying the poly U tract in the guide RNA. A poly U tract that can be destroyed may include a series of 4, 5, 6, 7, 8, 9, or 10 U's.

In general, the tracr mate sequence comprises: (1) excision of a guide sequence flanked by the tracr mate sequence in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at the target sequence (CRISPR complex Includes any sequence that has sufficient complementarity with the tracr sequence to promote one or more of the tracr mate sequences that are hybridized to the tracr sequence). In general, the degree of complementarity follows the optimal alignment of the tracr mate and tracr sequences along the shorter length of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm and may further describe self-complementarity within secondary structures, such as tracr sequences or tracr mate sequences. In some embodiments, the degree of complementarity between the tracr and tracr mate sequences along the two shorter lengths is about or about 25%, 30%, 40 when optimally aligned. %, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, the tracr sequence is about or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, Nucleotide lengths greater than 40, 50, or greater. In some embodiments, the tracr and tract mate sequences are contained within a single transcript so that hybridization between the two produces a transcript having a secondary structure, eg, a hairpin. In one embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has 2, 3, 4 or 5 hairpins. In a further embodiment of the invention, the transcript has at most 5 hairpins. In the hairpin structure, the last “N” and the portion of the sequence 5 ′ upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3 ′ to the loop corresponds to the tracr sequence. Further non-limiting examples of a single polynucleotide comprising a guide sequence, a tracr mate sequence, and a tracr sequence are listed below (listed 5 ′ to 3 ′), where “N” represents the base of the guide sequence: The first lowercase block represents the tracr mate sequence, the second lowercase block represents the tracr sequence, and the last poly T sequence represents the transcription terminator:
In some embodiments, sequences (1) to (3) are Used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a transcript that is separate from the transcript comprising the tracr mate sequence.

  Consideration of tracr mate sequences for orthologs: In some embodiments, recombinant templates are also provided. The recombinant template can be a component of another vector described herein, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, the recombination template is designed to function as a template in homologous recombination within or near the target sequence, for example, nicked or cleaved by the CRISPR enzyme as part of the CRISPR complex. Is done. The template polynucleotide can be of any suitable length, for example, about or about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. possible. In some embodiments, the template polynucleotide is complementary to the portion of the polynucleotide that includes the target sequence. When optimally aligned, the template polynucleotide can overlap with one or more nucleotides (eg, about or more than about 1, 5, 10, 15, 20, or more nucleotides) of the target sequence. . In some embodiments, when the polynucleotide comprising the template sequence and the target sequence is optimally aligned, the nearest nucleotide of the template polynucleotide is about 1, 5, 10, 15, 20, 25, 50 from the target sequence. , 75, 100, 200, 300, 400, 500, 1000, 5000, 10,000, or more nucleotides.

  In some embodiments, the CRISPR enzyme has one or more heterologous protein domains (eg, other about or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or other CRISPR enzymes). It is part of a fusion protein containing a larger number of domains). The CRISPR enzyme fusion protein can include any additional protein sequence and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, Protein domains having one or more of transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, Examples include green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, such as blue fluorescent protein (BFP). CRISPR enzymes bind to DNA molecules or to proteins or fragments of proteins that bind to other cellular molecules, such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding It can be fused to gene sequences encoding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of a fusion protein comprising a CRISPR enzyme are described in US Patent Application Publication No. 20110059502, which is incorporated herein by reference. In some embodiments, tagged CRISPR enzymes are used to identify the localization of the target sequence.

  In some embodiments, the CRISPR enzyme may form a component of an inducible system. The inductive nature of the system allows gene editing using energy forms or spatiotemporal control of gene expression. Examples of energy forms include, but are not limited to, electromagnetic radiation, acoustic energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (phytochrome, LOV domain, or crypto) Chromium). In some embodiments, the CRISPR enzyme can be part of a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence specific manner. The light component can include a CRISPR enzyme, a light-responsive cytochrome heterodimer (eg, from Arabidopsis thaliana), and a transcriptional activation / repression domain. Additional examples of inducible DNA binding proteins and methods of use thereof are described in US Provisional Patent Application No. 61 / 746,465 and US Provisional Patent Application No. 61 / 721,283, which are incorporated herein by reference in their entirety. Provided to.

  In some aspects, the invention provides for one or more polynucleotides, for example, or one or more vectors described herein, one or more transcripts thereof, and / or one or more transcribed therefrom. A method is provided that includes delivering a protein to a host cell. In some embodiments, the present invention further provides cells produced by such cells, and animals comprising or produced from such cells. In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to the cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer a nucleic acid encoding a component of the CRISPR system to cells in culture or into a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and delivery vehicles such as nucleic acids complexed with liposomes. Viral vector delivery systems include DNA and RNA viruses that have a genome that is episomal or integrated after delivery to a cell. For an overview of gene therapy procedures, see Anderson, Science 256: 808-813 (1992); Nabel & Felgner, TIBTECH 11: 211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 95 &P; , British Medical Bull tin 51 (1): 31-44 (1995); Haddada et al. , In Current Topics in Microbiology and Immunology, Doerfler and Boehm (eds) (1995); and Yu et al. , Gene Therapy 1: 13-26 (1994).

  Non-viral delivery methods for nucleic acids include lipofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. Can be mentioned. Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) Commercially available (eg, Transfectam ™ and Lipofectin ™). Suitable cations and neutral lipids for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or target tissues (eg, in vivo administration).

  The preparation of lipid: nucleic acid complexes, such as targeted liposomes, eg, immunolipid complexes, is well known to those skilled in the art (eg, Crystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Gene Therapy 2: 710-722 (1995); Ahmad et al., Cancer Res. 52: 4817-4820 (1992); U.S. Patent No. 4,186,183; No. 17,344, No. 4,235,871, No. 4,261,975, No. 4,485,054, No. 4,501,728 Nos. 4,774,085, 4,837,028, and 4,946,787).

  The use of RNA or DNA virus-based systems for the delivery of nucleic acids utilizes a highly evolved process that targets the virus to defined cells in the body and transports the viral payload to the nucleus. Viral vectors can be administered directly to a patient (in vivo), or they can be used to treat cells in vitro, and optionally modified cells can be administered to a patient (ex vivo). . Conventional virus-based systems can include retrovirus, lentivirus, adenovirus, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is considered for retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues.

  Retroviral tropism can be altered by incorporating foreign envelope proteins to expand the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Thus, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors contain cis-acting long terminal repeats with packaging capabilities for foreign sequences up to 6-10 kb. The minimal cis-acting LTR is sufficient for vector replication and packaging, which is then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV) based, or combinations thereof (See, for example, Buchscher et al., J. Virol. 66: 2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommerfeld et al., Virol. 176: 58-59 (1990); Wilson et al., J. Virol. 63: 2374-2378 (1989); Miller et al., J. Virol.65: 2220-2224 (1991); / See Pat. 05,700).

  In applications where transient expression is preferred, an adenovirus-based system can be used. Adenovirus-based vectors can exhibit very high transduction efficiency in many cell types and do not require cell division. For such vectors, high titers and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.

  For example, cells can be transduced with target nucleic acid using in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures using adeno-associated virus (“AAV”) vectors (eg, West et al. Virology 160: 38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5: 793-801 (1994); Muzyczka, J See Clin.Invest.94: 1351 (1994) The construction of recombinant AAV vectors has been described in numerous publications, including, for example, US Patent No. 5,173,414, Tratschin et al., Mol. Biol 5: 3251-3260 (1985); Tratschin, et al., Mol.Cell.Biol.4: 2072-2081 (1984); Hermonat & Muzyczka, PNAS 81: 6466-6470 (1984); and Samulski et al., J. Virol.63: 03822-3828 (1989).

  Typically, packaging cells are used to form viral particles that can infect host cells. Such cells include 293 cells that package adenovirus, and ψ2 cells or PA317 cells that package retroviruses. Viral vectors used in gene therapy are usually generated by producing cell lines that package nucleic acid vectors into viral particles. Vectors typically contain the minimal viral sequences required for packaging and subsequent integration into the host, with other viral sequences replaced by expression cassettes for the polynucleotide to be expressed. . Defective viral functions are typically supplied in trans by packaging cell lines. For example, AAV vectors used in gene therapy typically have only ITR sequences from the AAV genome that are required for packaging and integration into the host genome. Viral DNA is packaged in cell lines containing helper plasmids that encode other AAV genes, namely rep and cap, but lack the ITR sequences. Cell lines can also be infected with adenovirus as a helper. Helper virus facilitates replication of AAV vectors and expression of AAV genes from helper plasmids. Helper plasmids are not packaged in significant amounts due to the lack of ITR sequences. Contamination by adenovirus can be reduced, for example, by heat treatment where adenovirus is more sensitive than AAV. Additional methods of delivering nucleic acids to cells are known to those skilled in the art. See, for example, US Patent Publication No. 20030087817, which is incorporated herein by reference.

  AAV is therefore considered an ideal candidate for use as a transduction vector. Such AAV transduction vectors may contain sufficient cis-acting functions to replicate in the presence of adenovirus or herpesvirus or poxvirus (eg, vaccinia virus) helper functions provided in trans. Recombinant AAV (rAAV) can be used to carry foreign genes into cells of various lineages. In these vectors, the AAV cap and / or rep genes are deleted from the viral genome and replaced with a selected DNA segment. Current AAV vectors can accommodate inserted DNA of up to 4300 bases.

  There are many methods for making rAAV, and the present invention provides methods for preparing rAAV and rAAV. For example, one or more plasmids comprising or consisting essentially of the desired viral construct are transfected into AAV infected cells. In addition, a second or additional helper plasmid is cotransfected into these cells to provide the AAV rep and / or cap genes essential for replication and packaging of the recombinant viral construct. Under these conditions, AAV rep and / or cap proteins act in trans to stimulate replication and packaging of the rAAV construct. RAAV is harvested 2-3 days after transfection. Traditionally, rAAV is recovered from cells along with adenovirus. The contaminating adenovirus is then inactivated by heat treatment. In the present invention, rAAV is advantageously recovered from the cell supernatant rather than from the cell itself. Thus, in a first aspect, the present invention provides for preparing rAAV, and in addition to the foregoing, rAAV can be prepared by a method comprising or consisting essentially of: exogenous comprising DNA for expression Infecting susceptible cells with rAAV containing DNA and a helper virus (eg, a poxvirus such as adenovirus, herpes virus, vaccinia virus), wherein the rAAV lacks a functional cap and / or rep (And helper viruses (eg, poxviruses such as adenovirus, herpes virus, vaccinia virus, etc. provide cap and / or rev functions that are deficient in rAAV); or exogenous DNA, including expression DNA RAAV containing sensitive cells Transfecting said cells with a step wherein the recombinant is deficient in a functional cap and / or rep and a plasmid providing a cap and / or rep function that is deficient in rAAV. Or infecting susceptible cells with rAAV containing foreign DNA including expression DNA, wherein the recombinant is deficient in functional cap and / or rep, Providing deficient cap and / or rep function; or recombining exogenous DNA with AAV deficient in functional cap and / or rep so that the exogenous DNA is expressed by the recombinant Transfect sensitive cells with plasmids for insertion into the body and to provide rep and / or cap functions A Rukoto, therefore by transfection, functional cap and / or rep deficient contains exogenous DNA comprising the expression for DNA that rAAV is provided.

  The rAAV may be derived from AAV as described herein, advantageously with rAAV1, rAAV2, AAV5 or rAAV having a hybrid or capsid that may comprise AAV1, AAV2, AAV5 or any combination thereof. possible. The AAV of rAAV can be selected in relation to the cells targeted by rAAV; for example, for targeting brain or neuronal cells, AAV serotypes 1, 2, 5 or hybrid or capsid AAV1, AAV2, AAV5 or Any combination thereof can be selected; and AAV4 can be selected for targeting heart tissue and AAV8 can be selected for targeting liver tissue.

  In addition to 293 cells, other cells that can be used in the practice of the invention and the relative infectivity of specific AAV serotypes in vitro for those cells (Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) is as follows.

  The present invention relates to an exogenous nucleic acid molecule encoding a CRISPR (clustered equidistant short batch repeat) system, such as a promoter and a CRISPR-related (Cas) protein (putative nuclease or helicase protein), such as a nucleic acid molecule encoding Cas9. And a first cassette comprising or consisting essentially of a terminator, and a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and two or more comprising or consisting essentially of a terminator , Advantageously 5 cassettes (for example, each cassette is generally represented as promoter-gRNA1-terminator, promoter-gRNA2-, for example) in total (including the first cassette), up to the packaging size limit of the vector. Terminator. Comprising or consisting of a plurality of cassettes comprising or consisting of a promoter-gRNA (N) -terminator (where N is the insertable number that is the upper limit of the packaging size limit of the vector) An essentially rAAV, or two or more individual rAAV, each comprising one or more cassettes of the CRISPR system, eg, the first rAAV contains a promoter and a Cas, eg, Cas9 A first cassette comprising or consisting essentially of an encoding nucleic acid molecule and a terminator, and the second rAAV comprises a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator, or A plurality of four cassettes consisting essentially of each (for example, each cassette is generally a Expressed as motor-gRNA1-terminator, promoter-gRNA2-terminator ... promoter-gRNA (N) -terminator, where N is the insertable number which is the upper limit of the packaging size limit of the vector RAAV is provided. Since rAAV is a DNA virus, the nucleic acid molecule in the discussion herein for AAV or rAAV is advantageously DNA. The promoter is advantageously the human synapsin I promoter (hSyn) in some embodiments.

Two approaches for packaging Cas9-encoding nucleic acid molecules, eg, DNA, into viral vectors to mediate genomic modification in vivo are preferred:
To achieve NHEJ-mediated gene knockout:
Single viral vector:
Vector containing two or more expression cassettes:
Promoter-Cas9-encoding nucleic acid molecule-terminator promoter-gRNA1-terminator promoter-gRNA2-terminator promoter-gRNA (N) -terminator (up to vector size limit)
Double virus vector:
Vector 1 containing one expression cassette to drive Cas9 expression
Promoter-Cas9-encoding nucleic acid molecule-terminator Vector 2 containing another expression cassette for driving the expression of one or more guide RNAs
Promoter-gRNA1-terminator promoter-gRNA (N) -terminator (up to the size limit of the vector)

  To mediate homologous recombination repair. In addition to the single and double viral vector approaches described above, additional vectors are used to deliver homologous recombination repair templates.

Promoters used to drive the expression of Cas9 encoding nucleic acid molecules may include:
The AAV ITR can function as a promoter: this is advantageous in eliminating the need for additional promoter elements, which can take up space in the vector. Restricted release of additional space can be used to drive expression of additional elements (such as gRNA). Also, ITR activity is relatively weak and can therefore be used to reduce toxicity due to overexpression of Cas9.

  For ubiquitous expression, promoters: CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain can be used.

For brain expression, promoters: synapsin I for all neurons, CaMKII alpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. can be used.
An albumin promoter can be used for liver expression.
SP-B can be used for lung expression.
ICAM can be used for endothelial cells.
For hematopoietic cells, IFN beta or CD45 can be used.
OG-2 can be used for osteoblasts.
Promoters used to drive the guide RNA can include:
PolIII promoter, eg U6 or H1
Use of the Pol II promoter and intronic cassette to express gRNA

  With respect to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. AAV of AAV for the cells to be targeted can be selected; for example, AAV serotypes 1, 2, 5 or hybrids or capsids AAV1, AAV2, AAV5 or any combination thereof for brain or neuronal cell targeting AAV4 can be selected for cardiac tissue targeting. AAV8 is useful for delivery to the liver. The above promoters and vectors are individually preferred.

  Additional methods of delivering nucleic acids to cells are known to those skilled in the art. See, for example, US Patent Publication No. 20030087817, which is incorporated herein by reference.

  In some embodiments, the host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected as it occurs naturally in the subject. In some embodiments, the transfected cells are harvested from the subject. In some embodiments, the cells are derived from cells harvested from the subject, eg, cell lines. A wide range of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1 , PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2 , WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, BALB / 3T3 mouse embryo fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10 .1 Mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd. 3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2 , EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells , Ku812, KCL22, KG1, KYO1, LN Cap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONO-MAC6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cells Line, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP , Vero cells, WM39, WT-49, X63, YAC-1, YAR, and And their transgenic varieties. Cell lines are available from a variety of sources known to those of skill in the art (see, eg, American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain one or more vector-derived sequences. In some embodiments, the CRISPR complex is transiently transfected with a component of the CRISPR system described herein (eg, by transient transfection of one or more vectors, or transfection with RNA). Cells that have been modified through body activity are used to establish new cell lines containing cells that contain the modification but lack any other exogenous sequences. In some embodiments, one or more cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells. Used in the evaluation of test compounds.

  In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. Methods for producing transgenic plants and animals are known in the art and generally begin with, for example, the methods of cell transfection described herein.

  With recent advances in crop genomics, the ability to perform efficient and cost-effective gene editing and manipulation using the CRISPR-Cas system transforms such genomes for improved production and improved traits This allows for rapid selection and comparison of single and multiplexed genetic manipulations. In this regard, U.S. Patents and Publications: U.S. Patent No. 6,603,061-Agrobacterium-Mediated Plant Transformation Method; U.S. Patent No. 7,868,149-Plant Genome Sequences and US Therf Applications. Publication No. 2009/0100536-Transgenic Plants with Enhanced Agnostic Traits, the contents and disclosure of each of which is hereby incorporated by reference in its entirety. In the practice of the present invention, Morrell et al "Crop genomics: advancements and applications" Nat Rev Genet. The contents and disclosure of 2011 Dec 29; 13 (2): 85-96 are also incorporated herein by reference in their entirety. In an advantageous embodiment of the invention, microalgae are engineered using the CRISPR / Cas9 system (Example 7). Accordingly, references to animal cells herein may apply mutatis mutandis and also apply to plant cells unless otherwise apparent.

  In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell that can be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including microalgae) and modifying one or more cells. Culturing can be performed ex vivo at any stage. One or more cells can also be reintroduced into a non-human animal or plant (including microalgae). For cells that are reintroduced, it is particularly preferred that the cells are stem cells.

  In some embodiments, the method comprises binding a CRISPR complex to a target polynucleotide to cause cleavage of the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises A CRISPR enzyme is complexed with a guide sequence that is hybridized to a target sequence within the target polynucleotide, said guide sequence being then bound to a tracr mate sequence that hybridizes to the tracr sequence.

  In one aspect, the present invention provides a method of altering the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises binding a CRISPR complex to a polynucleotide such that the binding results in an increase or decrease in expression of the polynucleotide; the CRISPR complex comprises the target polynucleotide A CRISPR enzyme complexed with a guide sequence that is hybridized to a target sequence within the guide sequence, which is then bound to a tracr mate sequence that hybridizes to the tracr sequence. Similar considerations apply to methods of modifying a target polynucleotide as described above. Indeed, these sampling, culturing and reintroduction options apply throughout aspects of the present invention.

  In one aspect, the present invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. The elements can be provided individually or in combination and can be provided in any suitable container, such as a vial, bottle, or tube. In some embodiments, the kit includes instructions in one or more languages, eg, two or more languages.

  In some embodiments, the kit includes one or more reagents used in a process that utilizes one or more of the elements described herein. The reagent can be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. Reagents can be provided in a form that can be used in a particular assay or in a form that requires the addition of one or more other components prior to use (eg, a concentrated or lyophilized form). The buffer may be any buffer, such as, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and It can be a combination of In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector for operably linking the guide sequence and regulatory elements. In some embodiments, the kit comprises a homologous recombination template polynucleotide.

  In one aspect, the present invention provides a method of using one or more elements of the CRISPR system. The CRISPR complex of the present invention provides an effective means of modifying the target polynucleotide. The CRISPR complexes of the present invention have broad utility, for example, modification of the target polynucleotide (eg, deletion, insertion, translocation, inactivation, activation) in a large number of cell types. Thus, the CRISPR complexes of the present invention have a wide range of uses, for example in gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex includes a CRISPR enzyme complexed with a guide sequence that is hybridized to a target sequence within the target polynucleotide. The guide sequence is then linked to a tracr mate sequence that hybridizes to the tract sequence.

  In one embodiment, the present invention provides a method of cleaving a target polynucleotide. The method includes modifying the target polynucleotide using a CRISPR complex that binds to the target polynucleotide and causes cleavage of the target polynucleotide. Typically, a CRISPR complex of the invention creates a break (eg, a single or double stranded break) in a genomic sequence when introduced into a cell. For example, a disease gene in a cell can be cleaved using this method.

  The cleavage created by the CRISPR complex can be repaired by repair processes such as the error prone non-homologous end joining (NHEJ) pathway or high fidelity homologous recombination repair (HDR). During these repair processes, exogenous polynucleotide templates can be introduced into the genomic sequence. In some methods, the genomic sequence is modified using an HDR process. For example, an exogenous polynucleotide template containing the sequence to be integrated, flanked by upstream and downstream sequences, is introduced into the cell. The upstream and downstream sequences share sequence similarity with both sides of the integration site in the chromosome.

  If desired, the donor polynucleotide is a delivery vehicle such as DNA, eg DNA plasmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), viral vector, linear DNA fragment, PCR fragment, naked nucleic acid, or liposome or poloxamer. It may be a nucleic acid complexed with.

  An exogenous polynucleotide template includes a sequence to be integrated (eg, a mutated gene). The integrating sequence may be a sequence that is endogenous or foreign to the cell. Examples of sequences to be integrated include a polynucleotide encoding a protein or a non-coding RNA (eg, microRNA). Thus, the integrating sequence can be operably linked to one or more suitable control sequences. Alternatively, the integrated sequence can provide a regulatory function.

  Upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. An upstream sequence is a nucleic acid sequence that shares sequence similarity with the genomic sequence upstream of the target integration site. Similarly, a downstream sequence is a nucleic acid sequence that shares sequence similarity with a chromosomal sequence downstream of the target integration site. The upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target genomic sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target genomic sequence. In some methods, upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the target genomic sequence.

  The upstream or downstream sequence is about 20 bp to about 2500 bp, for example about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700. 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, exemplary upstream or downstream sequences have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more specifically about 700 bp to about 1000 bp.

  In some methods, the exogenous polynucleotide template can further include a marker. Such markers can facilitate screening for target integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide templates of the present invention can be constructed using recombinant techniques (see, eg, Sambrook et al., 2001 and Ausubel et al., 1996).

  In an exemplary method of modifying a target polynucleotide by integrating an exogenous polynucleotide template, a double-stranded break is introduced into the genomic sequence by the CRISPR complex, which cleavage is homologous recombination with the exogenous polynucleotide template. The template is repaired by being integrated into the genome. The presence of double strand breaks facilitates template integration.

  In other embodiments, the invention provides methods for altering the expression of polynucleotides in eukaryotic cells. The method includes increasing or decreasing the expression of the target polynucleotide using a CRISPR complex that binds to the polynucleotide.

  In some methods, the target polynucleotide can be inactivated to achieve altered expression in the cell. For example, when a CRISPR complex binds to a target sequence in a cell, the target polynucleotide is such that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function as the wild type sequence functions. Is inactivated. For example, the protein or microRNA coding sequence may be inactivated such that no protein is produced.

  In some methods, a control sequence can be inactivated so that it no longer functions as a control sequence. As used herein, “control sequence” refers to any nucleic acid sequence that provides for transcription, translation, or accessibility of a nucleic acid sequence. Examples of regulatory sequences include promoters, transcription terminators, and enhancers are regulatory sequences.

  An inactivated target sequence can be a deletion mutation (ie, deletion of one or more nucleotides), insertion mutation (ie, insertion of one or more nucleotides), or nonsense mutation (ie, stop codon). Substitution of another nucleotide with a single nucleotide such that is introduced). In some methods, inactivation of the target sequence results in a “knock out” of the target sequence.

  Changes in the expression of one or more genomic sequences associated with biochemical signaling pathways assay for differences in mRNA levels of the corresponding genes between test model cells and control cells when contacted with a candidate agent. Can be determined. Alternatively, differences in the expression of sequences associated with biochemical signaling pathways are determined by detecting differences in the level of the encoded polypeptide or gene product.

  To assay changes caused by agents at the level of mRNA transcripts or corresponding polynucleotides, the nucleic acid contained in the sample is first extracted according to standard methods in the art. For example, Sambrook et al. MRNA can be isolated using various lytic enzymes or chemical solutions according to the procedure shown in (1989), or extracted with nucleic acid binding resins according to the instructions provided by the manufacturer. The mRNA contained in the extracted nucleic acid sample is then subjected to amplification procedures or conventional hybridization assays (eg, Northern blot analysis) according to methods well known in the art or based on the methods exemplified herein. Detected.

  For the purposes of the present invention, amplification means any method that uses primers and polymerases that have the ability to replicate the target sequence with reasonable fidelity. Amplification can be performed by natural or recombinant DNA polymerases, such as Taq Gold ™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. A preferred amplification method is PCR. Specifically, the isolated RNA can be subjected to a reverse transcription assay combined with quantitative polymerase chain reaction (RT-PCR) to quantify the expression level of sequences associated with biochemical signaling pathways. .

  Detection of gene expression levels can be performed in real time during amplification assays. In one aspect, amplification products can be directly visualized with fluorescent DNA binding agents, such as, but not limited to, DNA intercalating agents and DNA groove binding agents. Because the amount of intercalating agent incorporated into the double-stranded DNA molecule is typically proportional to the amount of amplified DNA product, it is convenient to interpolate using conventional optical systems in the art. By quantifying the fluorescence of the calate dye, the amount of amplification product can be determined. Suitable DNA binding dyes for this application include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acriflavine, fluorescent coumarin, ellipticine, Daunomycin, chloroquine, distamycin D, chromomycin, fomidium, mitramycin, ruthenium polypyridyl, anthramycin and the like can be mentioned.

  In another aspect, other fluorescent labels, such as sequence specific probes, can be used in the amplification reaction to facilitate detection and quantification of amplification products. Probe-based quantitative amplification relies on sequence-specific detection of the desired amplification product. This amplification utilizes a fluorescent target-specific probe (eg, TaqMan® probe) that results in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well established in the art and are taught in US Pat. No. 5,210,015.

  In yet another aspect, conventional hybridization assays can be performed using hybridization probes that share sequence homology with sequences associated with biochemical signaling pathways. Typically, a probe is capable of forming a stable complex with a sequence associated with a biochemical signal transduction pathway contained in a biological sample obtained from a test subject by a hybridization reaction. It will be appreciated by those skilled in the art that when antisense is used as the probe nucleic acid, the target polynucleotide provided in the sample is selected to be complementary to the sequence of the antisense nucleic acid. Conversely, if the nucleotide probe is a sense nucleic acid, the target polynucleotide is selected to be complementary to the sequence of the sense nucleic acid.

  Hybridization can be performed under conditions of various stringencies, eg, as described herein. Preferred hybridization conditions for the practice of the present invention are those in which the recognition interaction between the probe and the sequence associated with the biochemical signaling pathway is sufficiently specific and sufficiently stable. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, for example, (Sambrook, et al., (1989); Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, second edition). Hybridization assays can be formed using probes immobilized on any solid support including, but not limited to, nitrocellulose, glass, silicon, and various gene arrays. A preferred hybridization assay is performed on a high density gene chip as described in US Pat. No. 5,445,934.

  In order to conveniently detect the probe-target complex formed during the hybridization assay, a nucleotide probe is conjugated to a detectable label. Detectable labels suitable for use in the present invention include any composition that is detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means. A wide variety of suitable detectable labels are known in the art, including fluorescent or chemiluminescent labels, radioisotope labels, enzymes or other ligands. In preferred embodiments, it may be desirable to use fluorescent labels or enzyme tags, such as digoxigenin, β-galactosidase, urease, alkaline phosphatase or peroxidase, avidin / biotin complex.

  The detection method used to detect or quantify the hybridization intensity will typically depend on the label selected above. For example, the radiolabel can be detected using photographic film or a phosphorimager. Fluorescent markers can be detected and quantified using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing a substrate to the enzyme and measuring the reaction product produced by the action of the enzyme on the substrate; and finally, the colorimetric label is simply a colored label. Is detected by visualizing.

  Changes in the expression of sequences associated with biochemical signaling pathways caused by the drug can also be determined by examining the corresponding gene product. For determination of protein levels, typically a) contacting a protein contained in a biological sample with an agent that specifically binds to a protein associated with a biochemical signaling pathway; and (b) The identification of any drug: protein complex formed in this way is involved. In one aspect of this embodiment, the agent that specifically binds to a protein associated with a biochemical signaling pathway is an antibody, preferably a monoclonal antibody.

  The reaction is a protein associated with a biochemical signaling pathway obtained from a test sample under conditions that allow a complex to form between the drug and the protein associated with the biochemical signaling pathway. This is performed by bringing a drug into contact with the sample. Complex formation can be detected directly or indirectly according to standard procedures in the art. In a direct detection method, the drug is provided with a detectable label and unreacted drug can be removed from the complex; thus, the amount of label remaining indicates the amount of complex formed. For such a method, it is preferable to select a label that remains bound to the drug even under stringent washing conditions. The label preferably does not interfere with the binding reaction. Alternatively, indirect detection procedures may use agents that contain a label introduced chemically or enzymatically. Desirable labels generally do not interfere with the binding or stability of the resulting drug: polypeptide complex. However, the label is typically designed to be accessible to the antibody for effective binding and thus the production of a detectable signal.

  A wide variety of labels suitable for protein level detection are known in the art. Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.

  The amount of drug: polypeptide complex formed during the binding reaction can be quantified by standard quantitative assays. As explained above, drug: polypeptide complex formation can be directly measured by the amount of label remaining at the binding site. In the alternative, a protein associated with a biochemical signaling pathway is tested for its ability to compete with a labeled analog for a binding site on a particular drug. In this competitive assay, the amount of label captured is inversely proportional to the amount of protein sequence associated with the biochemical signaling pathway present in the test sample.

  Many protein analysis techniques based on the general principles outlined above are available in the art. This includes, but is not limited to, radioimmunoassay, ELISA (enzyme linked immunoradiometric assay), “sandwich” immunoassay, immunoradiometric assay, in situ immunoassay (eg, colloidal gold, enzyme or radioisotope labeling) ), Western blot analysis, immunoprecipitation assay, immunofluorescence assay, and SDS-PAGE.

  For performing the protein analysis described above, antibodies that specifically recognize or bind to proteins associated with biochemical signaling pathways are preferred. If desired, antibodies that recognize certain types of post-translational modifications (eg, biochemical signaling pathway induced modifications) can be used. Post-translational modifications include but are not limited to glycosylation, lipidation, acetylation, and phosphorylation. These antibodies may be purchased from commercial suppliers. For example, anti-phosphotyrosine antibodies that specifically recognize tyrosine phosphorylated proteins are available from a number of suppliers, including Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodies are particularly useful in detecting proteins that are otherwise phosphorylated at their tyrosine residues in response to ER stress. Such proteins include, but are not limited to, eukaryotic translation initiation factor 2α (eIF-2α). Alternatively, these antibodies can be made by immunizing a host animal or antibody producing cell with a target protein that exhibits the desired post-translational modification using conventional polyclonal or monoclonal antibody techniques.

  In practicing the subject method, it may be desirable to identify expression patterns of proteins associated with biochemical signaling pathways in different body tissues, different cell types, and / or different intracellular structures. Such tests may be performed using tissue-specific, cell-specific or intracellular structure-specific antibodies that have the ability to bind protein markers that are preferentially expressed in specific tissues, cell types, or intracellular structures. it can.

  Changes in the expression of genes associated with biochemical signaling pathways can also be determined by examining changes in the activity of the gene product relative to control cells. Assays for changes in the activity of proteins associated with biochemical signaling pathways caused by the drug may depend on the biological activity and / or signaling pathway being investigated. For example, if the protein is a kinase, changes in its ability to phosphorylate one or more downstream substrates can be determined by various assays known in the art. Exemplary assays include, but are not limited to, immunoblots and immunoprecipitations with antibodies such as anti-phosphotyrosine antibodies that recognize phosphorylated proteins. In addition, kinase activity is measured by high-throughput chemiluminescence assays such as AlphaScreen ™ (available from Perkin Elmer) and eTag ™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111: 162-174). Can be detected.

  A pH-sensitive molecule such as a fluorescent pH dye can be used as a reporter molecule when the protein associated with the biochemical signaling pathway is part of a signaling cascade that results in fluctuations in intracellular pH conditions. In another example where the protein associated with the biochemical signaling pathway is an ion channel, changes in membrane potential and / or intracellular ion concentration can be monitored. Many commercial kits and high-throughput devices are particularly suitable for rapid and robust screening for ion channel modulators. Representative instruments include FLIPR ™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences). These instruments have the ability to detect reactions simultaneously in over 1000 sample wells on a microplate and provide real-time measurements and functional data within 1 second or even 1 millisecond.

  In performing any of the methods disclosed herein, without limitation, microinjection, electroporation, sonoporation, particle bombardment, calcium phosphate mediated transfection, cationic transfection, liposome transfection, dendrimer trait Transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, nucleic acid uptake enhanced by targeted drugs, and liposomes, immunoliposomes, virosomes, Alternatively, suitable vectors can be introduced into cells or embryos by one or more methods known in the art, including delivery via artificial virions. In some methods, the vector is introduced into the embryo by microinjection. One or more vectors can be microinjected into the nucleus or cytoplasm of the embryo. In some methods, one or more vectors can be introduced into a cell by nucleofection.

  The target polynucleotide of the CRISPR complex can be any polynucleotide that is endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide that remains in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence that encodes a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA).

  Examples of target polynucleotides include sequences associated with biochemical signaling pathways, such as biochemical signaling pathway related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide is any gene that produces a transcriptional or translational product in an abnormal level or in an abnormal form in a cell derived from an affected tissue compared to a non-disease control tissue or cell Or refers to a polynucleotide. It may be a gene that becomes abnormally high expressed; it may be a gene that becomes abnormally low expressed, where the change in expression correlates with disease development and / or progression To do. A disease-related gene also refers to a gene that has one or more mutations or genetic variations that are directly involved in the cause of the disease or in linkage disequilibrium with one or more genes involved. The transcribed or translated product may be known or unknown and may be at a normal or abnormal level.

  The target polynucleotide of the CRISPR complex can be any polynucleotide that is endogenous or foreign to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence that encodes a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA). Without being bound by theory, it is thought that the target sequence should be associated with a PAM (protospacer flanking motif); ie, a short sequence recognized by the CRISPR complex. The exact sequence and length requirements for PAM will vary depending on the CRISPR enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the protospacer (ie, target sequence). is there. Examples of PAM sequences are listed in the Examples section below and one skilled in the art can identify additional PAM sequences that are used for a given CRISPR enzyme.

  The target polynucleotides of the CRISPR complex include US provisional patent application No. Broad reference number BI-2011 / 008 / WSGR reference number 44063-701.101 and BI-2011 / 008 / WSGR reference number 44063-701.102, respectively. 61 / 736,527 and 61 / 748,427 (both filed on the title SYSTEMS METHODS AND COMPOSTIONS FOR SEQUENCE MANIPULATION, December 12, 2012 and January 2, 2013, respectively) The entire contents of which are incorporated herein by reference in their entirety) and a number of disease-related genes and polynucleotides and signaling biochemical pathway-related genes and polynucleotides listed in Plastid can be mentioned.

  Examples of target polynucleotides include sequences associated with signaling biochemical pathways, such as signaling biochemical pathway associated genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide is any gene that produces a transcriptional or translational product at an abnormal level or in an abnormal form in cells derived from the affected tissue compared to a non-disease control tissue or cell Or refers to a polynucleotide. This can be a gene that becomes expressed at an abnormally high level; it can be a gene that becomes expressed at an abnormally low level, and the change in expression is the development and / or progression of the disease Correlate with A disease-related gene also refers to a gene having a mutation or genetic variation that is directly responsible for the pathogenesis of the disease or in linkage disequilibrium with the gene responsible for the pathogenesis of the disease. The product to be transcribed or translated can be known or unknown, and can be at a normal or abnormal level.

  Examples of disease-related genes and polynucleotides are McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (available from Baltimore, Med.), And National Center for Biotechnology, National Center for Biotechnology. Available from the World Wide Web.

  Examples of disease-related genes and polynucleotides are listed in Tables A and B. Disease-specific information is available from McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Med.) And National Center for Biotechnology Information from National Center for Biotechnology Information. It is available. Examples of signaling biochemical pathway related genes and polynucleotides are listed in Table C.

  Mutations in these genes and pathways can result in the production of inappropriate amounts of inappropriate protein or proteins that affect function. Additional examples of genes, diseases and proteins are described in US Provisional Patent Application No. 61 / 736,527, filed December 12, 2012, and 61/748, filed January 2, 2013. No. 427 is incorporated herein by reference. Such genes, proteins and pathways can be target polynucleotides of the CRISPR complex.

  Embodiments of the invention also relate to methods and compositions related to gene knockout, gene amplification, and repair of specific mutations associated with DNA repeat instability and neurological disease (Robert D. Wells, Tetsuo). (Ashizawa, Genetic Institutes and Neurological Diseases, Second Edition, Academic Press, Oct 13, 2011-Medical). It has been found that tandem repeat sequences of defined aspects are responsible for more than 20 human diseases (New insights into repeat instability: role of RNA and DNA hybrids. McIvor EI, Polak U, Napierala M. RNA RNA. -Oct; 7 (5): 551-8). These abnormalities of genomic instability can be corrected using the CRISPR-Cas system.

  A further aspect of the invention relates to the use of the CRISPR-Cas system for correcting abnormalities in the EMP2A and EMP2B genes that have been identified as being associated with Lafora disease. Lafora's disease is an autosomal recessive condition characterized by progressive myoclonic epilepsy that can begin as an epileptic seizure in adolescence. Several cases of this disease can be caused by mutations in genes that have not yet been identified. The disease causes seizures, muscle spasms, difficulty walking, dementia, and ultimately death. Currently, there are no treatments that have proven effective against disease progression. Other genetic abnormalities associated with epilepsy can also be targeted by the CRISPR-Cas system, and the underlying genetics are described in Genetics of Epilepsy and Genetic Epilepsies, Giuliano Avanzani, Jeffrey L., et al. Edited by Noebels, further described in Mariani Foundation Paidatory Neurology: 20; 2009).

  In still another embodiment of the present invention, the CRISPR-Cas system is used, and Genetic Diseases of the Eye, Second Edition, and edited by Elias. Ocular abnormalities resulting from several gene mutations described further in Traboursi, Oxford University Press, 2012 can be corrected.

  Some further aspects of the present invention are the broader described further under the topic subsection Genetic Disorders on the National Institutes of Health website (website at health.nih.gov/topic/GeneticDisorders). It relates to correction of abnormalities related to genetic diseases. Examples of genetic brain diseases include, but are not limited to, adrenoleukodystrophy, corpus callosum deficiency, Aicardy syndrome, Alpers disease, Alzheimer's disease, Bath syndrome, Batten disease, CADASIL, cerebellar degeneration, Fabry disease, Gerstmann- Streisler-Scheinker disease, Huntington's disease and other triplet repeat diseases, Leigh disease, Lesch-Nyhan syndrome, Menkes disease, mitochondrial myopathy and NINDS colpocephaly. These diseases are further described under the subsection Genetic Brain Disorders on the National Institutes of Health website.

  In some embodiments, the condition is neoplasia. In some embodiments where the pathology can be neoplasia, the gene to be targeted can be any of those listed in Table A (in this case, PTEN, etc.). In some embodiments, the condition can be age related macular degeneration. In some embodiments, the condition can be schizophrenia. In some embodiments, the condition can be a trinucleotide repeat disorder. In some embodiments, the condition can be fragile X syndrome. In some embodiments, the condition can be a secretase-related disorder. In some embodiments, the condition can be a prion-related disorder. In some embodiments, the condition can be ALS. In some embodiments, the condition can be drug preference. In some embodiments, the condition can be autism. In some embodiments, the condition can be Alzheimer's disease. In some embodiments, the condition can be inflammation. In some embodiments, the condition can be Parkinson's disease.

  Examples of proteins associated with Parkinson's disease include, but are not limited to, α-synuclein, DJ-1, LRRK2, PINK1, parkin, UCHL1, symphrin-1, and NURR1.

  Examples of preference-related proteins include ABAT.

  Examples of inflammation-related proteins include, for example, monocyte chemotactic protein-1 (MCP1) encoded by the Ccr2 gene, CC chemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, and Fcgr2b gene And IgG receptor IIB (also referred to as FCGR2b, CD32), or Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene.

  Examples of cardiovascular disease-related proteins include IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin I2 (prostacyclin) synthase), MB (myoglobin) ), IL4 (interleukin 4), ANGPT1 (Angiopoietin 1), ABCG8 (ATP binding cassette, subfamily G (WHITE), member 8), or CTSK (cathepsin K).

  Examples of Alzheimer's disease-related proteins include, for example, the very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or the UBA3 gene The NEDD8 activating enzyme E1 catalytic subunit protein (UBE1C) encoded by

  Examples of proteins associated with autism spectrum disorders include, for example, benzodiazepine receptor (peripheral) -related protein 1 (BZRAP1) encoded by the BZRAP1 gene, AF4 encoded by the AFF2 gene (also referred to as MFR2) / FMR2 family member 2 protein (AFF2), vulnerable X mental retardation autologous homolog 1 protein (FXR1) encoded by FXR1 gene, or vulnerable X mental retardation autosomal homolog 2 protein (FXR2) encoded by FXR2 gene Can be mentioned.

  Examples of proteins associated with macular degeneration include, for example, ATP binding cassette subfamily A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, apolipoprotein E protein (APOE) encoded by the APOE gene, Another example is a chemokine (CC motif) ligand 2 protein (CCL2) encoded by the CCL2 gene.

  Examples of proteins associated with schizophrenia include NRG1, ErbB4, CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

  Examples of proteins involved in tumor suppression include, for example, ATM (capillary telangiectasia ataxia mutation), ATR (capillary telangiectasia ataxia and Rad3-related), EGFR (epidermal growth factor receptor), ERBB2 ( v-erb-b2 erythroblastic leukemia virus oncogene homolog 2), ERBB3 (v-erb-b2 erythroblastic leukemia virus oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia virus cancer) A gene homolog 4), Notch1, Notch2, Notch3, or Notch4 can be mentioned.

  Examples of proteins associated with secretase disorders include, for example, PSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor Body protein), APH1B (anterior pharyngeal defect 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer's disease 4)), or BACE1 (beta site APP cleavage enzyme 1). .

  Examples of proteins related to amyotrophic lateral sclerosis include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein) , VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

  Examples of proteins related to prion diseases include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelium) Mention may be made of growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof.

  Examples of proteins related to neurodegenerative conditions in prion disorders include, for example, A2M (alpha-2-macroglobulin), AATF (apoptotic antagonist transcription factor), ACPP (prostatic acid phosphatase), ACTA2 (aortic smooth muscle actin alpha 2) ), ADAM22 (ADAM metallopeptidase domain), ADORA3 (adenosine A3 receptor), or ADRA1D (alpha-1D adrenergic receptor for alpha-1D adrenergic receptor).

  Examples of proteins associated with immunodeficiency include, for example, A2M [alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase]; ABCA1 [ATP binding cassette subfamily A (ABC1), member 1] ABCA2 [ATP binding cassette subfamily A (ABC1), member 2]; or ABCA3 [ATP binding cassette subfamily A (ABC1), member 3].

  Examples of proteins associated with trinucleotide repeat disorders include, for example, AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (Huntington), or DMPK (Myotonic dystrophy protein kinase), FXN (Frataxin), ATXN2 (Ataxin 2) are mentioned.

  Examples of proteins related to neurotransmission disorders include, for example, SST (somatostatin), NOS1 (nitric oxide synthase 1 (neural type)), ADRA2A (adrenergic alpha-2A receptor), ADRA2C (adrenergic alpha) -2C receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-hydroxytryptamine (serotonin) receptor 2C).

  Examples of neurodevelopment-related sequences include, for example, A2BP1 [Ataxin 2 binding protein 1], AADAT [amino adipate aminotransferase], AANAT [arylalkylamine N-acetyltransferase], ABAT [4-aminobutyric acid aminotransferase], ABCA1 [ATP binding cassette subfamily A (ABC1) member 1] or ABCA13 [ATP binding cassette subfamily A (ABC1) member 13].

  Further examples of preferred conditions that can be treated by the system of the present invention can be selected from the following: Alcardi-Gutierre syndrome; Alexander disease; Alan-Herndon-Dudley syndrome; POLG-related disorders; alpha-mannosidosis (Type II and type III); Alstrem syndrome; Angelman syndrome; telangiectasia ataxia; neuronal ceroid lipofuscinosis; beta-serasamia; bilateral optic atrophy and (infant type) optic atrophy 1 type; retinoblast (Bilateral); Canavan disease; brain / eye / face / skeletal syndrome 1 [COFS1]; cerebral tendon xanthomatosis; Cornelia de Lang syndrome; MAPT-related disorder; hereditary prion disease; Drave syndrome; Familial Alzheimer's disease; Friedreich's ataxia [FRDA]; Fucosidosis; Fukuyama congenital muscular dystrophy; Galactosialidosis; Gaucher's disease; Organic acidemia; Hemophagocytic lymphohistiocytosis; Hutchinson-Gilford progeria syndrome; Jerber Lange-Nielsen syndrome; zygote epidermolysis bullosa; Huntington's disease; Krabbe disease (infant type); Mitochondrial DNA-related Leigh syndrome and NARP; Lesch-Nyhan syndrome; LIS1-related synovial dysfunction; Low syndrome; MECP2 duplication syndrome; ATP7A-related copper transport disorder; LAMA2-related muscular dystrophy; arylsulfatase A deficiency; mucopolysaccharidosis type I, II or III; peroxisome dysplasia, Zellweger syndrome Neurodegeneration with brain iron accumulation disease; acid sphingomyelinase deficiency; Niemann-Pick disease type C; glycine encephalopathy; ARX-related disorder; urea cycle disorder; COL1A1 / 2-related osteogenesis disorder; Deletion syndrome; PLP1-related disorder; Perry syndrome; Ferran-Mcdermot syndrome; glycogen storage type II (Pompe disease) (infant type); MAPT-related disorder; MECP2-related disorder; limb-type punctate cartilage dysplasia type 1; Roberts syndrome; Sandhoff disease; Schindler disease type 1; adenosine deaminase deficiency; Smith-Lemli-Opitz syndrome; spinal muscular atrophy; early childhood-onset spinocerebellar ataxia; Collagen type VI related disorder; Usher syndrome type I; congenital muscular dystrophy; Wolf -Hirschhorn syndrome; lysosomal acid lipase deficiency; and xeroderma pigmentosum.

  As will be apparent, it is envisioned that the system of the present invention can be used to target any desired polynucleotide sequence. Some examples of conditions or diseases that can be usefully treated using the system of the present invention are included in the table above, and examples of genes currently associated with those conditions are also provided in the table. However, the exemplified genes are not exclusive.

  The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any manner. This example, along with the methods described herein, is a representative example of a presently preferred embodiment, is exemplary, and is not intended to limit the scope of the invention. Variations and other uses within the spirit of the invention as defined by the scope of the claims will be made by those skilled in the art.

Example 1: Improvement of the Cas9 system for in vivo use Applicants performed a metagenomic search for Cas9 with a small molecular weight. Most Cas9 orthologs are quite large. Many known Cas9 orthologs are huge and contain more than 1300 amino acids. For example, SpCas9 is approximately 1368 amino acids long, which is too large to be packaged into a viral vector for delivery. A graph representing the length distribution of the Cas9 homolog is shown in FIG. The graph is created from the sequence deposited with GenBank. Some arrays are annotated incorrectly, so the exact frequency for each length is not always correct. Nevertheless, this gives an overview of the distribution of Cas9 protein, suggesting the presence of a shorter Cas9 homolog.

  Applicants have found by computer analysis that there are two Cas9 proteins of less than 1000 amino acids in the bacterial strain Campylobacter. The sequence of one Cas9 from Campylobacter jejuni is provided below. At this length, CjCas9 can be easily packaged into AAV, lentivirus, adenovirus, and other viral vectors for robust delivery in vivo to primary cells and in animal models. In a preferred embodiment of the invention, Cas9 protein from S. aureus is used.

Campylobacter jejuni Cas9 (CjCas9)

The putative tracrRNA element for this CjCas9 is:

The direct repeat sequence is:

An example of a chimeric guide RNA for CjCas9 is the following:

Example 2: Cas9 diversity and chimeric RNA
The wild type CRISPR-Cas system is an adaptive immune mechanism against invading exogenous DNA used by diverse species across bacteria and archaea. The type II CRISPR-Cas system consists of a set of genes that encode proteins responsible for “acquisition” of foreign DNA into the CRISPR locus, and a set of genes that encode “executions” of the DNA cleavage mechanism; DNA nuclease (Cas9), non-coding trans-activated crRNA (tracrRNA), and an array of foreign DNA-derived spacers (crRNA) flanked by direct repeats. Upon maturation by Cas9, the tracRNA and crRNA duplexes guide the Cas9 nuclease to the target DNA sequence defined by the spacer guide sequence, are required for cleavage, and are short in the target DNA specific for the respective CRISPR-Cas system. Mediates double-strand breaks in DNA near strand sequence motifs. The type II CRISPR-Cas system has been found throughout the bacterial kingdom, and the Cas9 protein sequence and size, tracrRNA and crRNA direct repeat sequences, the genomic organization of those elements, and the motif requirements for targeted cleavage are highly diverse It is. Certain species may have multiple distinct CRISPR-Cas systems.

  Applicants are based on sequence homology with known Cas9 and known subdomains, such as the HNH endonuclease domain and the RuvC endonuclease domain [information from Eugene Koonin and Kira Makalova] and orthologous structures. From the identified bacterial species, 207 putative Cas9s were evaluated. Phylogenetic analysis based on this set of protein sequence conservation revealed five families of Cas9, including three groups of large Cas9 (approximately 1400 amino acids) and two groups of small Cas9 (approximately 1100 amino acids) (Figure 4 and 5A-F).

  In some embodiments, the tracr mate sequence or direct repeat is downloaded from the CRISPR database, or They are identified in silico by searching for repeated motifs found in a 2 kb window of the genomic sequence flanking the type II CRISPR locus, ranging from 2.20 to 50 bp and every 3.20 to 50 bp. In some embodiments, two of these criteria may be used, for example, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used. In some embodiments, the candidate tracrRNA is subsequently 1. 1. Sequence homology with direct repeats (motif search in Geneious with up to 18 bp mismatch), 2. 2. the presence of a predicted Rho-independent transcription terminator in the direction of transcription, and Predicted by stable hairpin secondary structure between tracrRNA and direct repeats. In some embodiments, two of these criteria may be used, for example, 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria can be used. In some embodiments, the chimeric synthesis guide RNA (sgRNA) design incorporates a double-stranded structure of at least 12 bp between the direct repeat and the tracrRNA.

  A list of human codon optimized Cas9 ortholog sequences for pairing with the chimeric RNA provided in FIGS. 8A-J is provided in FIGS. Applicants have also shown that Cas9 orthologs can cleave their targets in an in vitro cleavage assay (FIG. 16). The CRISPR loci in some of these families are shown in FIG. The corresponding guide RNA sequence is shown in FIG. Applicants systematically analyzed genomic DNA sequences within about 2 kb of Cas9 protein using custom computational analysis codes and identified direct repeats ranging from 35 bp to 50 bp with intervening spacers ranging from 29 bp to 35 bp. . Based on the direct repeat sequence, Applicants searched for a tracRNA candidate sequence having the following criteria: computationally outside the crRNA array, but containing a high degree of homology with the direct repeat (direct repeat: required for tracrRNA base pairing; custom computational analysis), outside of the coding region of the protein component, containing a Rho-independent transcription termination signal approximately 60 bp to 120 bp downstream from the region of homology from the direct repeat Cofolding with direct repeats to form a duplex, followed by two or more hairpin structures at the distal end of the tracrRNA sequence. Based on these predictive criteria, Applicants selected 18 Cas9 proteins and their uniquely related direct repeats and an initial set of tracrRNAs distributed across all five Cas9 families. Applicants have conserved the sequence and secondary structure of the natural direct repeat: tracrRNA duplex, while shortening the region that base pairs and fuses two RNA elements through an artificial loop. Further sets were generated (Figures 8A-J).

Example 3: Cas9 orthologs Applicants generated codon optimized Cas9 orthologs for improved expression in eukaryotic cells.

An example of a human codon optimized sequence (ie optimized for expression in humans) sequence: SaCas9 is provided below.

  Applicants analyzed the Cas9 ortholog to identify the relevant PAM sequence and the corresponding chimeric guide RNA as shown in FIGS. This expanded set of PAMs provides broader targeting across the genome, significantly increases the number of unique target sites, and offers the potential to identify novel Cas9 with increased levels of specificity in the genome. Applicants have determined that the PAM for Staphylococcus aureus subspecies Aureus Cas9 is NNGRR (FIG. 14). Staphylococcus aureus subspecies Aureus Cas9 is also known as SaCas9. Figures 15a-d provide single or multiple vector designs of SaCas9.

  FIG. 7 shows the sequence logo for the putative PAM indicated by the reverse complementary strand. Cas9 orthologs and their respective sgRNAs were used to cleave libraries of targets carrying randomized PAMs (7 bp sequences flanking the target sequence 3 '). The cleavage product was isolated and deeply sequenced to generate a 7 bp candidate sequence that allowed cleavage for each Cas9 ortholog. For each Cas9 ortholog, consensus PAM was determined by aligning all 7 bp candidate sequences (FIGS. 7 and 21).

  While further experiments testing the thermodynamics and in vivo stability of sgRNA-DNA duplexes are likely to generate additional predictive power for off-target activity, the development of SpCas9 mutants and orthologs is improved. New variants with different specificities can be generated. The specificity of the Cas9 ortholog can be further assessed by testing the ability of each Cas9 to tolerate a mismatch between the guide RNA and its DNA target.

Example 4: Cas9 Mutation In this example, Applicants show that the following mutations can convert SpCas9 to a nicking enzyme: D10A, E762A, H840A, N854A, N863A, D986A.

  Applicants provide sequences that indicate where the mutation point is located within the SpCas9 gene (FIGS. 10A-M). Applicants also show that nickase can still mediate homologous recombination. Furthermore, we show that SpCas9 with these mutations (individually) reduces the level of double-strand breaks. All Cas9 orthologs share the general organization of 3-4 RuvC and HNH domains (FIG. 19). The 5'-most RuvC domain cleaves the non-complementary strand and the HNH domain cleaves the complementary strand. All notations refer to guide sequences.

  The catalytic residue of the 5′RuvC domain is linked to the target Cas9 with other Cas9 orthologs (S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. pneumoniae). Mutating the conserved Asp residue to alanine, identified by homology comparison with S. thermophilus CRISPR locus 3 and Franciscilla novicida type II CRISPR locus To convert Cas9 into a complementary strand nicking enzyme. Similarly, Cas9 is converted to a non-complementary strand nicking enzyme by mutating the conserved His and Asn residues of the HNH domain to alanine.

Example 5: Cas9 function optimization For enhancement or development of new functions, Applicants generated chimeric Cas9 protein by combining fragments from different Cas9 orthologs.

For example, Applicants fused the N-terminus of St1Cas9 (fragment from this protein is shown in bold) with the C-terminus of SpCas9 (fragment from this protein is underlined).
> St1 (N) Sp (C) Cas9
> Sp (N) St1 (C) Cas9

  Applicants also generated Sp_St3 chimeric proteins, indicating in vitro cleavage by SpCas9, St3Cas9, Sp_St3 chimeras and St3_Sp chimeras (FIG. 17).

Benefits of making chimeric Cas9 include the following:
a. Toxicity reduction b. Improved expression in eukaryotic cells c. Increased specificity d. Reduction of protein molecular weight, production of smaller proteins by combining minimal domains from different Cas9 homologs.
e. Changes to PAM array requirements

Example 6: In vivo Cas9 delivery using AAV particles or vectors In vivo delivery-AAV method AAV has advantages over other viral vectors for several reasons:
Low toxicity (this may be due to purification methods that do not require ultracentrifugation of cell particles that can activate the immune response)
-The probability of causing insertional mutagenesis is low because it does not integrate into the host genome.

  Some current AAV vectors can accommodate up to 4300 bases of inserted DNA as upper or packaging limits, while AAV can have 4.5 or 4.75 KB of inserted DNA. This means that the DNA encoding Cas9 enzyme and the promoter and transcription terminator must all be compatible with the same viral vector. Constructs larger than 4.5 or 4.75 KB result in significantly reduced virus production. SpCas9 is completely large and the gene itself exceeds 4.1 kb, which makes it difficult to pack into AAV. Thus, embodiments of the present invention include utilizing shorter Cas9 orthologs. For example:

  FIG. 3 provides a schematic representation of AAV vectors that can be used in the methods and compositions of the invention. Packaging is discussed above.

Example 7: Method of delivering microalgal engineering Cas9 using Cas9

  Method 1: Applicants deliver Cas9 and guide RNA using a vector that expresses Cas9 under the control of a constitutive promoter such as Hsp70A-Rbc S2 or beta2-tubulin.

  Method 2: Applicants deliver Cas9 and T7 polymerase using a vector that expresses Cas9 and T7 polymerase under the control of a constitutive promoter such as Hsp70A-Rbc S2 or beta2-tubulin. Guide RNA is delivered using a vector containing a T7 promoter that drives the guide RNA.

  Method 3: Applicants deliver Cas9 mRNA and in vitro transcribed guide RNA to algal cells. RNA can be transcribed in vitro. Cas9 mRNA consists of the coding region for Cas9 and the 3'UTR from Copl to ensure the stabilization of Cas9 mRNA.

  For homologous recombination, Applicants provide additional homologous recombination repair templates.

Cassette for driving Cas9 expression under the control of the beta-2 tubulin promoter, followed by the sequence for the Cop1 3′UTR.

The cassette that drives the expression of T7 polymerase under the control of the beta-2 tubulin promoter, followed by the sequence for the Cop1 3′UTR:

Sequence of guide RNA driven by T7 promoter (T7 promoter, N represents target sequence):

Gene delivery:
Chlamydomonas reinhardtii strains CC-124 and CC-125 from Chlamydomonas Resource Center are used for electroporation. The electroporation protocol follows the standard recommended protocol from the GeneArt Chlamydomonas Engineering kit (website information at tools.invitrogen.com/content/sfs/manuals/genet_chlamy_kits_man.pdf).

  Applicants also generate a line of Chlamydomonas reinhardtii that constitutively expresses Cas9. This can be done using pChlamy1 (linearized using PvuI) and selecting hygromycin resistant colonies. The sequence for pChlammy1 containing Cas9 is shown below. In this approach to achieve gene knockout, it is only necessary to deliver RNA for the guide RNA. For homologous recombination, Applicants deliver guide RNA and linearized homologous recombination template.

pChlamy1-Cas9:

  For all modified Chlamydomonas reinhardtii cells, Applicants have confirmed good modification using PCR, SURVEYOR nuclease assay, and DNA sequencing.

Example 8: SaCas9 and PAM recognition for in vivo applications Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) CRISPR / CasPR as functional genomic engineering tools in mammalian cells After identification, Applicants initiated this project to seek further utilization of the diversity of the Type II CRISPR / Cas system.

By defining a new functional type II CRISPR / Cas system for use in mammalian cells, Applicants can potentially find the following:
(1) CRISPR / Cas system with higher efficiency and / or specificity (2) CRISPR / Cas system with different protospacer adjacent motifs (PAM) allowing targeting of a wider range of genomic loci (3) CRISPR / Cas systems with smaller sizes (thus the applicants have their packaging size limit in vivo in a single vector (current Sp or St systems exceed this limit of 4.7 kb) ) Can be delivered by a mammalian viral delivery system, such as an adeno-associated virus (AAV) vector)
And other desired traits.

  Identification and design of the Sa CRISPR / Cas system for in vivo applications. Applicants tested a new type II CRISPR / Cas system in Staphylococcus aureus (Sa) that functions in vitro in a dsDNA cleavage assay and identified a putative PAM for NNGRRT. A component of this system is a guide CRISPR RNA with a direct repeat (DR) from Sa that forms a functional guide RNA complex with the Cas9 protein from Sa and the tracrRNA from Sa. This ternary system is similar to all other type II CRISPR / Cas systems. Accordingly, Applicants have designed a two-component system, and Applicants have developed a short-chain stem-as we have done for the Streptococcus pyogenes (Sp) CRISPR / Cas system. Sa tracRNA was fused to Sa guide CRISPR RNA via a loop to form a chimeric guide RNA. This chimeric guide RNA could support the cleavage of dsDNA in vitro. Therefore, Applicants decided to clone the complete binary system: cas9 and chimeric guide RNA into AAV vectors to test their functionality in living organisms.

  Applicants selected the AAV system. This is because it has a broad tropism in different tissues / organs depending on the serotype that has been shown to be safe for in vivo applications, and also supports long-term expression of transgenes in living organisms, This is because it is a non-integrating ssDNA-based non-immunogenic mammalian virus.

  The initial AAV vector design has (1) a CMV promoter that drives a SaCas9 protein with a single NLS and HA epitope tag, and (2) a human U6 promoter that drives a chimeric RNA. These are placed between two inverted terminal repeats (ITRs) from the most well studied AAV serotype 2 that function as viral packaging signals.

  The PAM sequence test on the endogenous mammalian genome is as follows: SaCas9 target spacer was selected across multiple genes to cover different potential PAM sequences. Different spacers are cloned into a U6-sgRNA (single guide RNA) expressing dsDNA cassette and the U6-sgRNA expressing dsDNA cassette is co-transformed into a mammalian cell line (293FT for human targets, N2a and Hepa for mouse targets) Introduced. 72 hours after transfection, all genomic DNA was extracted and subjected to a surveyor nuclease assay. Run through a TBE Page gel to detect genomic cleavage. Quantify and plot the genomic DNA cleavage efficiency.

  Summary of genome cleavage efficiency and other statistics for all test targets

  Summary of genome cleavage efficiency and other statistics for all test targets (cleanup version)

  The results from the PAM test are shown in FIGS. Comprehensive testing of over 100 targets identified that the PAM for SaCas9 can be described as NNGRR (not the NNGRRT mentioned above).

  PAM test summary: (1) NNGRR for generic SaCas9PAM useful for new target design, (2) Double nickase test with new target, (3) NNGRG may be a more powerful PAM .

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  While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now make numerous variations, modifications, and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used in the practice of the invention.

Claims (20)

A composition comprising a CRISPR complex comprising:
The CRISPR complex is
I. A CRISPR-Cas polynucleotide sequence (there may be more than one),
(A) an engineered guide sequence comprising RNA, capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of the CRISPR complex to the target sequence;
(B) a tracr mate sequence comprising RNA and capable of hybridizing to a tracr sequence, and (c) a CRISPR-Cas-based polynucleotide sequence comprising a tracr sequence comprising RNA, and II. A type II Cas9 protein from Staphylococcus aureus, wherein the type II Cas9 protein comprises one or more nuclear localization sequences (NLS), comprising a type II Cas9 protein,
Composition. The tracr mate sequence hybridizes to the tracr sequence, the guide sequence directs sequence specific binding of the CRISPR complex to a target sequence;
The CRISPR complex is the type II Cas9 protein, which is complexed with (1) the guide sequence hybridized to the target sequence and (2) the tracr mate sequence hybridized to the tracr sequence. Comprising the type II Cas9 protein,
The composition of claim 1.   The composition according to claim 1, wherein the CRISPR-Cas polynucleotide sequence is a chimeric RNA (chiRNA).   4. The composition according to any one of claims 1 to 3, comprising nanoparticles, liposomes, exosomes, yeast systems or microvesicles. A composition comprising a vector system comprising one or more vectors, wherein the one or more vectors comprise the following CRISPR complex components:
I. A CRISPR-Cas polynucleotide sequence (there may be more than one),
(A) an engineered guide sequence comprising RNA, capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of the CRISPR complex to the target sequence;
(B) a tracr mate sequence comprising RNA and capable of hybridizing to a tracr sequence, and (c) a CRISPR-Cas-based polynucleotide sequence comprising a tracr sequence comprising RNA, and II. A type II Cas9 protein derived from Staphylococcus aureus, wherein said type II Cas9 protein comprises a polynucleotide sequence encoding a type II Cas9 protein comprising one or more nuclear localization sequences (NLS) A composition comprising.   6. The composition of claim 5, wherein the one or more vectors comprise one or more viral vectors.   7. The composition of claim 6, wherein the one or more viral vectors comprise one or more retrovirus, lentivirus, adenovirus, adeno-associated virus or herpes simplex virus vector.   The composition according to any one of claims 5 to 7, wherein the composition comprises a single vector.   9. The composition according to any one of claims 5 to 8, wherein the polynucleotide sequence encoding the type II Cas9 protein is codon optimized for expression in eukaryotic cells.   10. The composition of any one of claims 5-9, wherein the tissue specific promoter directs expression of the CRISPR complex component in muscle, neuron, bone, skin, blood, liver, pancreas or lymphocyte.   11. The composition according to any one of claims 1 to 10, wherein the tracr sequence has a length of 30 or more nucleotides.   12. A composition according to any one of the preceding claims, wherein the tracr sequence has a length of 50 or more nucleotides.   13. The composition according to any one of claims 1 to 12, wherein the type II Cas9 protein comprises two or more nuclear localization sequences (NLS).   The composition according to claim 1, wherein the composition is for genome engineering.   An ex vivo or in vitro host cell or cell line comprising a composition according to any one of claims 1 to 14, wherein the host cell or cell line is not a human germline cell. Or cell line.   16. An ex vivo or in vitro host cell or cell line according to claim 15, which is a stem cell or stem cell line.   15. A method of modifying a eukaryote by manipulation of one or more target sequences at a genomic locus of interest, said method comprising applying to the organism a composition according to any one of claims 1-14. Delivering, wherein the eukaryote is a non-human organism.   15. An ex vivo method of modifying a eukaryotic cell by manipulation of one or more target sequences at a genomic locus of interest, wherein the method comprises the composition according to any one of claims 1-14. Delivering to the cell, wherein the method does not comprise a process for altering the genetic identity of the human germline.   The method according to claim 17 or 18, wherein the organism is a plant.   15. Use of a composition according to any one of claims 1-14 in ex vivo gene or genome editing, said use not comprising a process for modifying the genetic identity of the human germline, Use is not a method for the treatment or prevention of a disease in the human body, the use does not include administering the system to the human body.